Modeling T-Cell Acute Lymphoblastic Leukemia Induced by the SCL and LMO1 Oncogenes

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Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes

Mathieu Tremblay,1,7 Ce´dric S. Tremblay,1,7 Sabine Herblot,1,8 Peter D. Aplan,2 JoseéHe´bert,3 Claude Perreault,1 and Trang Hoang1,4,5,6,9 1Institute of Research in Immunology and Cancer, University of Montreal, Montreál, Que´bec H3C 3J7, Canada; 2Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA; 3Banque de Cellules Leuce´miques du Que´bec, Maisonneuve-Rosemont Hospital, Montreál, Que´bec H1T 2M4, Canada; 4Department of Pharmacology, Faculty of Medicine, University of Montreál, Montreál, Que´bec H3C 3J7, Canada; 5Department of Biochemistry, Faculty of Medicine, University of Montreál, Montreál, Que´bec H3C 3J7, Canada; 6Department of Molecular Biology, Faculty of Medicine, University of Montreál, Montreál, Que´bec H3C 3J7, Canada

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. Here we show that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrb clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes. [Keywords: T-ALL; Notch1; SCL/TAL1; LMO1; leukemia-initiating cell; pre-TCR] Supplemental material is available at http://www.genesdev.org. Received December 18, 2009; revised version accepted April 12, 2010.

Studies of human and murine cancers have identified genes encoding oncogenic transcription factors (Look 1997), multigenic hits that cooperate in cell transformation (Berns which are ‘‘master’’ gene regulatory proteins (Rabbitts 1988; Hahn and Weinberg 2002). However, deciphering the 1991) in hematopoiesis. These observations suggest that multistep process that transforms normal cells into fully the process of cell transformation subverts cell fate deter- metastazing cancer cells remains a challenge. Strategies minants. Recent genome-wide approaches based on gene aimed at dissecting cancer genes indicate that they con- expression profiling, analysis of copy number alterations, verge to enhance self-renewal, favor growth factor inde- array CGH, or second-generation deep-sequencing technol- pendence, and cause differentiation arrest (Jonkers and ogy have identified a number of deletions, new mutations, Berns 1996; Hanahan and Weinberg 2000). Interestingly, or dysregulated gene expression in T-ALL (for review, see 70% of recurring chromosomal translocations in child- Mullighan and Downing 2009). These powerful approaches hood T-cell acute lymphoblastic leukemia (T-ALL) involve nonetheless likely overlook active, but unmutated, signaling pathways that are essential for the viability of transformed cells (Luo et al. 2009). 7 These authors contributed equally to this work. Elevated expression of the SCL/TAL1 transcription fac- 8Present address: Pediatrics Department, Faculty of Medicine, University of Montreal, and Charles-Bruneau Cancer Centre, Sainte-Justine Hospital, tor and/or its nuclear partner, LMO1 or LMO2, is found in Montre´al, Que´bec H3T 1C5, Canada 50%–60% of childhood T-ALL, associated with gene rear- 9Corresponding author. E-MAIL [email protected]; FAX (514) 343-6945. rangements (Begley et al. 1989; Boehm et al. 1991) or not Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1897910. (Ferrando et al. 2002; for review, see Aifantis et al. 2008).

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SCL is a member of the basic helix–loop–helix (bHLH) progenitor arrested at the same stage as the bulk of tumor family of transcription factors that specifies the hemato- cells. However, oncogenes can modify cellular pheno- poietic fate from mesodermal precursors, whereas LMO types, as observed in several transgenic mouse models in proteins are zinc-binding LIM domain proteins (for review, which the DN-to-DP progression can occur in the ab- see Lecuyer and Hoang 2004). Inappropriate SCL expres- sence of pre-TCR signaling due to ectopic expression of sion in the thymus of SIL-SCL, CD2-SCL,orSca1-SCL BCL2 and NOTCH1/3 or inhibition of E-protein function transgenic mice (SCLtg) impairs T-cell differentiation (Linette et al. 1994; Engel and Murre 2002; Michie et al. (Larson et al. 1996; Herblot et al. 2000) during the preleu- 2007). Furthermore, evidence for the presence of a sub- kemic stage without inducing leukemia (Aplan et al. 1997; population of leukemia-initiating cells (LICs) with stem Curtis et al. 1997; Goardon et al. 2002), whereas Lck-SCL cell properties in T-ALL remains to be documented. transgenic mice develop T-cell lymphomas with low pen- In the present study, we investigated the cellular and mo- etrance (Condorelli et al. 1996; Kelliher et al. 1996). lecular events in SCL–LMO1-induced T-ALL using trans- LMO1tg or LMO2tg mice occasionally develop T-ALL after genic mice as a model. a long latency period of ;18 mo (Larson et al. 1994). SCL collaborates with LMO1 (Aplan et al. 1997) or LMO2 (Larson et al. 1996) to accelerate disease onset (3–6 mo) Results and confer full penetrance. While SCL–LMO1/2 are suffi- The SCL and LMO1 oncogenes expand early thymic cient to initiate leukemogenesis in transgenic mice, other precursor (ETP)/DN1 thymocytes events are required for emergence of full-blown leukemia, as evidenced by a highly variable latency period. Circulating bone marrow-derived progenitors (Kit+Sca1+LinÀ Gain-of-function mutations of the NOTCH1 gene are [KSL]) settle in the thymus, where they give rise to ETPs, found in >50% of human T-ALL (Pear and Aster 2004; a subset of DN1 cells with high T-lineage potential Weng et al. 2004) and in SCLtgLMO1tg mouse models of (Sambandam et al. 2005), endowed with the highest pro- T-ALL (Lin et al. 2006; O’Neil et al. 2006; Gothert et al. liferative potential in the thymus. ETPs further differenti- 2007). Members of the NOTCH family are evolutionary ate into DN2, DN3, and DN4 cells (Fig. 1A). While tran- conserved proteins that control cell fate. NOTCH1 signal- sition from the ETP to DN2 and DN3 stages is associated ing regulates the T-cell versus B-cell fate choice in pre- with 10-fold expansion in cell numbers at each step, at the thymic progenitors (Sambandam et al. 2005; Aifantis et al. DN4-to-DP transition, thymocytes actively cycle under 2008), and also acts at later stages of T-cell development the influence of the pre-TCR, resulting in an ;200-fold (Zweidler-McKay and Pear 2004). NOTCH1 cooperates expansion of cell numbers (Fig. 1A; Supplemental Fig. S1A) with E2A (Ikawa et al. 2006) and the pre-T-cell antigen when cells acquire the CD4 and CD8 markers (DP) as receptor (pre-TCR) (Ciofani et al. 2004; Aifantis et al. 2008) well as TCRa/b expression (Fig. 1A). To assess the earliest to specify T-lineage commitment. In the thymus, the pre- cell types that are affected by the oncogenes, we first TCR (for review, see von Boehmer et al. 1999; Aifantis analyzed progenitors in the bone marrow and thymus of et al. 2008) controls the critical transition from the SCLtgLMO1tg mice during the preleukemic stage; i.e., at CD4ÀCD8À double-negative (DN) to the double-positive 3–4 wk after birth. The SCL transgene is driven by the SIL (DP; CD4+CD8+) stages, and coordinates cell proliferation, promoter, which is active in all proliferating cells (Supple- cell survival, and differentiation in the a/b lineage. In mental Fig. S1D), whereas LMO1 expression is driven by leukemogenesis, the contribution of the pre-TCR appears the Lck transgenic cassette. Expression levels of the variable, as it is dispensable in some mouse models (Liao endogenous Lck and the LMO1 transgene were assessed et al. 1998; Engel and Murre 2002) but important in others by RT–PCR. Both were absent in bone marrow KSL, but (Bellavia et al. 2002; dos Santos et al. 2007). In the latter, expression was initiated in thymic ETP/DN1 cells and was the precise contribution of the pre-TCR to the leukemo- found at higher levels in all subsequent thymocyte subsets genic process remains unknown. Finally, the importance (Figs. 1A; Supplemental Fig. S1B,C), indicating that the Lck of the pre-TCR was reported as minor in SCL–LMO1- transgenic cassette is active in all thymic precursors, but dependent leukemias (Fasseu et al. 2007), although SCL inactive in bone marrow progenitors. Accordingly, bone expression in childhood T-ALL is invariably associated marrow KSL numbers were not affected by the transgenes with high TCR expression (Ferrando et al. 2002). Further- (Supplemental Fig. S2A). In the thymus, however, the SCL– more, SCL-dependent leukemias in mice harbor in-frame LMO1 transgenes induced a 10-fold expansion of the ETP TCRb rearrangements (Chervinsky et al. 2001). Hence, the population compared with wild-type mice, whereas the pre-TCR or TCR pathway may be relevant to the patho- variations induced by each transgene separately were not physiology of SCL-associated T-ALL. significant (Fig. 1B). This expansion was followed by an Cell transformation results from the accumulation of expansion of DN3/DN4 populations (Fig. 1C), whereas the multiple genetic anomalies (Hahn and Weinberg 2002), DP population was decreased, consistent with previous arguing for the likelihood that the initiating event occurs reports indicating that SCL and LMO1 inhibit thymocyte in a cell with extended proliferative potential, allowing differentiation (Larson et al. 1996; Aplan et al. 1997; for the acquisition of additional mutations. However, the Herblot et al. 2000). Consistent with decreased DP thymo- cell of origin of leukemia need not be a normal stem cell cytes, total cell numbers in the thymus of SCL–LMO1 trans- (for review, see Jordan 2009). It was originally thought genic mice were decreased compared with age-matched that ALLs could originate from a committed lymphoid wild-type mice. Thus, the SCL–LMO1 transgenes induce

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Notch1 interacts with SCL and LMO1 in T-ALL

Figure 1. The SCL and LMO1 oncogenes expand the population of ETPs and induce CD3-dependent but antigen-independent T-ALL. (A) Schematic diagram of thymocyte populations and their cell surface markers. ETPs are Kit+Sca1+LinÀCD25À. Thymocyte populations (DN1–DN4, DP, SP4, and SP8) are Thy1.2+ and can be further differentiated on the basis of CD25, CD44, CD4, and CD8 expression. The pre-TCR is essential for DN-to-DP transition, whereas the TCR regulates negative and positive selection. Shown are the absolute numbers, the percentages of cells in S/G2/M phase, and Lck expression levels as assessed by real-time PCR for each subset of wild-type thymocyte. (B) The absolute number of ETPs was assessed by flow cytometry analysis of wild-type, SCLtg, LMO1tg,andSCLtgLMO1tg mice at the preleukemic stage (3 wk old). Shown are box plots delimited by the lower and upper 25 percentiles of the distribution with results from Student’s t-test ([*] P < 0.001 compared with wild type). The lineinsidetheboxplotsrepresentsthemedian, and those outside represent the two extreme values. (C) The absolute number of DN1-to-DP populations was assessed by flow cytometry analysis in wild-type and preleukemic SCLtgLMO1tg mice as in B.(D) Diagram of the transplantation strategy. Indicated leukemic populations were purified from Rag1+/+SCLtgLMO1tg thymomas and transplanted into immunodeficient Rag1À/À mice at concentrations of 103 (DN and DP) or 104 (DN1, DN3, DN4, ISP8 and DP) cells. Examples of tumor phenotypes from primary and secondary leukemias, as well as Kaplan-Meier curves of the time to leukemia, are shown.

an expansion of DN thymocytes, starting from the ETP, plantation of 106 leukemic cells induced T-ALL in all and inhibit the DN–DP transition. secondary hosts (data not shown). There was, however, a significant difference among the two groups of mice re- LICs are enriched in the DN population ceiving 103 leukemic cells. By 12 wk after transplantation, almost all mice transplanted with DN leukemic cells were At time of overt leukemia, the leukemic population is moribund, whereas almost all mice grafted with DP leu- heterogeneous and comprises DN and DP cells, as reported kemic cells remained healthy during this time frame (Fig. (Larson et al. 1994; Kelliher et al. 1996; Chervinsky et al. 1D). Furthermore, both leukemic DN and DP populations 1999). Leukemia is sustained by self-renewing LICs that induced secondary leukemias that reproduced the pheno- can be detected by transplantation into unirradiated im- type of the primary tumor in transplanted hosts (Fig. 1D). munodeficient mice (Hope et al. 2004). Leukemias arising Therefore, despite an apparent DN–DP hierarchy based on in SCLtgLMO1tg mice were analyzed for their capacities cell surface markers, LICs are found in both DN and DP to initiate secondary leukemias in Rag1À/À mice. We rea- populations. The leukemia-initiating potential of the DN soned that the same clonal marker should appear in pri- population was nonetheless higher than that of the DP pop- mary and secondary leukemias, as an indication of self- ulation, and was found mostly in the DN3 and DN4 renewal. Since the rearrangement of the Tcrb locus is a populations (Fig. 1D). Even though the preleukemic DN1 marker of clonality in Tcells and of SCL–LMO1 leukemias population was expanded, the leukemic DN1 population (Chervinsky et al. 1999), these leukemias were analyzed lacks leukemia-initiating potential. for Vb8 and Vb5 rearrangement by PCR prior to and after transplantation (Supplemental Fig. S3). We found the same pre-TCR deficiency delays leukemia onset rearrangement in primary and secondary leukemias, in- in SCLtgLMO1tg mice dicating that the same clone has undergone self-renewal. We next assessed the presence of LIC in leukemic DN and The DN3-to-DP transition, which is affected by the DP populations purified from primary leukemias. Trans- transgenes during the preleukemic phase, is controlled

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Tremblay et al. by the pre-TCR. To address the importance of the pre- We took an independent genetic approach with Rag1- TCR in cell transformation, we exploited two genetic deficient mice in which T-cell differentiation is arrested models in which thymocyte differentiation is blocked at at the DN3 stage, because Rag1À/À cells are unable to re- the DN3 stage; i.e., Cd3eÀ/À and Rag1À/À mice. arrange their Tcr genes and therefore do not express the The pre-TCR and TCR require association with the CD3 pre-TCR, the TCRa/b,orTCRg/d. Rag1-deficient mice complex for signaling (Malissen et al. 1995); therefore, expressing the SCL–LMO1 oncogenes developed leukemia Cd3-deficient cells have nonfunctional pre-TCR/TCRs. with a similar kinetic as Cd3eÀ/ÀSCLtgLMO1tg mice (Fig. SCLtgLMO1tg mice develop T-ALL with 100% penetrance 2A). Of note, the survival curves for Rag1À/ÀSCLtgLMO1tg (Fig. 2A), as reported (Aplan et al. 1997). However, on and Cd3eÀ/ÀSCLtgLMO1tg mice are almost superimpos- a Cd3eÀ/À background, the penetrance of the disease was able. Together, our observations concur to indicate the decreased by 48% (Figs. 2A–4F) and the median survival importance of pre-TCR signaling as a collaborating event increased by 234 d when compared with Cd3e+/+ litter- with the SCL and LMO1 oncogenes. mates. Our results indicate a strong genetic interaction between the SCL–LMO1 oncogenes and Cd3e, but differ to Major histocompatibility complex (MHC)-mediated some extent with the report by Fasseu et al. (2007) showing antigen presentation and leukemia onset a complete abrogation of SCL–LMO1-induced leukemo- in SCLtgLMO1tg mice genesis in Cd3eÀ/À mice. This discrepancy is currently unresolved, but could be due to differences in genetic Signaling by the pre-TCR and the TCR is important for backgrounds. All of our mice have been backcrossed on thymocyte survival and proliferation at the DN and DP a C57BL6 background for >10 generations, excluding the stages, respectively. The pre-TCR is antigen-independent possibility of modifier loci introduced by genetic crossing (Irving et al. 1998; Gibbons et al. 2001), whereas the ac- between Cd3eÀ/À and SCLtgLMO1tg mice. Together, these tivation of TCR signaling is dependentoneitherMHCclass observations indicate that pre-TCR/TCR signaling collab- I or MHC class II antigen presentation by antigen-presenting orates with the SCL–LMO1 oncogenes to accelerate and cells (APCs) (Matzinger and Bevan 1977; Marrack and increase the penetrance of leukemia. Kappler 1988). Differentiation arrest at the DN3 stage in

Figure 2. Leukemia initiation is reduced in the absence of CD3/pre-TCR signaling. (A) Kaplan-Meier curves of the time to leukemia for SCLtgLMO1tg, Cd3eÀ/À SCLtgLMO1tg, Rag1À/ÀSCLtgLMO1tg, and AbbÀ/ÀB2mÀ/À SCLtgLMO1tg mice. All malignant thymic lymphomas were diagnosed at necropsy. The median survival times for each cohort of n mice are shown. (B) Preleukemic and leukemic phenotypes of SCLtgLMO1tg thymocytes. Representative flow cytometry analysis of thymocytes from wild-type and Cd3eÀ/À SCLtgLMO1tg as well as nontransgenic control littermates was performed during the preleukemic stage (4 wk old) and at the time of overt leukemia. Numbers in gray are for preleukemic cells and numbers in black are for overt leukemia phenotype. (C) The absolute number of DN and DP populations was assessed by flow cytometry analysis for Cd3e+/+ SCLtgLMO1tg and Cd3eÀ/ÀSCLtgLMO1tg tumors. Box plots shown as in Figure 1B ([*] P < 0.05 as compared with Cd3e+/+SCLtgLMO1tg mice). (D) Time to leukemia after transplantation of 106 leukemic Cd3e+/+ SCLtgLMO1tg or Cd3eÀ/À SCLtgLMO1tg cells in Rag1À/À mice (n mice per group). Shown are the median survival times for each cohort. (E) Representative leukemic phenotypes of primary and secondary Cd3e+/+ and Cd3eÀ/À SCLtgLMO1tg tumors as in B.(F) Kaplan-Meier curves of the time to leukemia for purified DN or DP leukemic populations from Cd3eÀ/ÀSCLtgLMO1tg thymomas transplanted into immunodeficient Rag1À/À mice at a concentration of 104.DataareshownasinFigure1D.

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Notch1 interacts with SCL and LMO1 in T-ALL

Rag1À/À and Cd3eÀ/À mice did not allow for an assessment transplantation. These observations indicate that Cd3e+/+ of the importance of the TCR in leukemogenesis, which is SCLtgLMO1tg leukemias have a higher potential to initiate required later at the DP stage. To directly address the leukemia in secondary hosts. functional importance of the TCR, we examined SCL– Despite the preponderance of the DP population, we LMO1-dependent T-ALL in mice deficient for both the noticed the presence of a minor population of DN cells in H2-Ab1 gene (Abb) and the b2-microglobulin gene Cd3eÀ/ÀSCLtgLMO1tg leukemias (Fig. 2B,C). We there- (b2m)—i.e., lacking MHC class I and class II (Grusby et al. fore purified DN and DP cells from these leukemias and 1993)—and therefore deficient for antigen presentation. found that DN leukemic cells have higher leukemia- Unlike Cd3eÀ/À mice, the kinetics of SCL–LMO1-induced initiating potential than DP leukemic cells (Fig. 2F). leukemia in AbbÀ/Àb2mÀ/À mice was not affected when Furthermore, both DN and DP cells fully reconstitute compared with wild-type littermates (Fig. 2A), despite im- the phenotypic composition of the primary tumor, giving paired differentiation at the DP stage (Supplemental Fig. rise to DP (70%), DN (4%), and immature single positive S4A), presumably due to defective antigen presentation 8 (ISP8; 15%) cells in transplanted hosts (Supplemental and, hence, defective TCR function. Finally, the pheno- Fig. S4C). type of these leukemia resemble those of Abb+/+b2m+/+ SCLtgLMO1tg mice (Supplemental Fig. S4A). The collabo- Cosegregation between Cd3 and Notch1 mutations ration between CD3 and the SCL–LMO1 oncogenes is in SCL-dependent leukemogenesis therefore antigen-independent and, hence, due to pre- TCR signaling. Our observations indicate that antigen- This high capacity to initiate secondary T-ALL in Cd3e+/+ dependent TCR signaling has no discernible contribution SCLtgLMO1tg leukemias suggests that additional genetic to leukemogenesis when the pre-TCR is functional, al- events might confer stem cell-like properties to popula- though we do not exclude the possibility that TCR signal- tions that are expanded by the SCL–LMO1 oncogenes. ing could be a collaborating event when the pre-TCR is Cytogenetic analysis of tumors from SCLtgLMO1tg mice by dysfunctional; for example, in the absence of pTa (Fasseu G-banding and SKY (Supplemental Figs. S5, S6) revealed et al. 2007). that three out of four tumors exhibit a normal karyotype. One out of four tumors showed the presence of trisomy 7 and/or 16. Thus, the SCL–LMO1 oncogenes did not cause Cosegregation between leukemia-initiating potential major chromosomal instability. Nonetheless, the clonal and Cd3 expression mark of rearranged Vb5 and Vb8 loci in primary leukemias To gain further insights into interactions between CD3 that was also found in secondary leukemias indicated that and SCL–LMO1, we analyzed thymocyte subsets in pre- the disease is monoclonal, regardless of Cd3 gene expres- leukemic and leukemic Cd3eÀ/ÀSCLtgLMO1tg mice. sion (Supplemental Fig. S3). These observations suggest DP cells were absent in Cd3eÀ/À (Fig. 2B) and Rag1À/À that one or a few clones acquired a proliferative advantage, (Supplemental Fig. S4A) mice, as expected, due to the lack possibly through the occurrence of additional mutations of pre-TCR signaling. Despite this, transgenic expression that were not detected by cytogenetic analysis. of SCL–LMO1 in Rag1À/À and Cd3eÀ/À mice induced the NOTCH1 gain-of-function mutations are frequently appearance of a minor population of DP cells (2%–5%) in found in T-ALL, mostly in the PEST domain and the the thymus during the preleukemic stage, and a dramatic heterodimerization domain (Weng et al. 2004; Roy et al. accumulation of DP cells in leukemic mice. Thus, the SCL 2007; Aifantis et al. 2008). Furthermore, NOTCH signals and LMO1 transgenes allowed for progression to the DP are required for the generation of ETPs (Sambandam et al. stage independently of the pre-TCR, indicating that the 2005), which we show here to be expanded by the SCL– phenotype of leukemic cells in this T-ALL does not reflect LMO1 oncogenes, and to sustain the growth of LICs in the developmental stage of the cell of origin of leukemia. human T-ALL (Armstrong et al. 2009). We therefore sys- Furthermore, the overwhelming progression to the DP tematically sequenced the Notch1 proto-oncogene using stage at the time of overt leukemia suggests that a sub- cDNAs amplified from six SCLtgLMO1tg leukemias aris- population of DN3 transgenic thymocytes has bypassed ing in Cd3e+/+ mice and seven in Cd3eÀ/À mice. We cov- the DN–DP checkpoint. Surprisingly, leukemic DN pop- ered sequences upstream of the heterodimerization do- ulations were expanded in Cd3e+/+ but not in Cd3eÀ/À main through to the 39 untranslated region (UTR) in tumors, whereas DP populations were more significantly three amplicons (Fig. 3A). Frame-shift insertions and expanded in Cd3eÀ/À tumors (Fig. 2B,C). In both geno- mutations that disrupt the PEST domain of Notch1 and types, these DP cells are, however, nonfunctional due to that are associated with a premature stop were found in the absence of CD3 or of low levels of TCRb (data not Cd3e+/+ leukemias, except for sample #241, consistent shown). with published reports (Fig. 3A; Lin et al. 2006; O’Neil To assess the presence of LICs in Cd3e+/+ and Cd3eÀ/À et al. 2006). No other mutation was found outside the leukemias, we transplanted 106 leukemic cells in adult PEST domain, including the heterodimerization domain. Rag1À/À mice (Fig. 2D,E). Mice grafted with Cd3e+/+ In contrast, no mutation was detected in seven different SCLtgLMO1tg leukemic cells reproducibly developed leu- Cd3eÀ/À leukemias induced by SCL–LMO1. The cosegre- kemia within a narrow window of 24–32 d. In sharp gation between Cd3 and Notch1 gain-of-function muta- contrast, 35% of mice transplanted with Cd3eÀ/À tions suggests a functional link between Notch1 and Cd3 SCLtgLMO1tg leukemic cells remained healthy 75 d after in T-ALL.

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Figure 3. Activating Notch1 mutations and high NOTCH1 protein levels in SCL– LMO1 T-ALL are dependent on Cd3 gene status. (A) The Notch1 gene from Cd3e+/+ SCLtgLMO1tg and Cd3eÀ/À SCLtgLMO1tg tumors was sequenced in three overlapping amplicons. Shown are the positions of the primers used in the sequencing reactions. HD, TAD, and PEST represent the heterodimerization, transactivation, and protein degradation domains, respectively. (B) NOTCH1 intracellular staining in Cd3e+/+ SCLtgLMO1tg and Cd3eÀ/ÀSCLtgLMO1tg primary tumor samples. The average fluo- rescence intensity per pixel from Z-stack confocal analysis from two Cd3e+/+SCLtg LMO1tg and three Cd3eÀ/ÀSCLtgLMO1tg different tumor samples is shown as mean 6 SD. (C) Quantitative RT–PCR of the indicated NOTCH1 target genes were performed in Cd3e+/+SCLtgLMO1tg and Cd3eÀ/À SCLtgLMO1tg tumors. Relative mRNA expression levels in tumor cells are normal- ized by CT values obtained with Cd3e+/+ SCLtgLMO1tg cells and are presented as box plots with the median and extreme values of each distribution ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001 as compared with Cd3eÀ/ÀSCLtgLMO1tg tumors). (D) The Notch1 gene from purified late preleukemic and leukemic thymocyte subsets from Cd3e+/+ SCLtgLMO1tg mice was sequenced as in A. (ISP8 corresponds to Thy1.2+CD8+CD4ÀCd3elo, SP4 corresponds to Thy1.2+CD4+CD8À, and SP8 corresponds to Thy1.2+CD4ÀCD8+.)

Cosegregation between Cd3 expression, NOTCH1 et al. 2002; Ong et al. 2006; Vacca et al. 2006), the elevated protein levels, and NOTCH1 activity levels of Notch3 in Cd3eÀ/À leukemias may compensate for in leukemic thymocytes lower NOTCH1 levels in these leukemias. Of note, Myc is expressed in both Cd3e+/+ and Cd3eÀ/À SCLtgLMO1tg To verify the consequences of mutations in the PEST do- tumors at levels comparable with Notch1tg tumors, consis- main, we first determined NOTCH1 protein levels by im- tent with an important role for c-Myc in leukemogenesis munofluorescence in Cd3e+/+ and Cd3eÀ/À SCL–LMO1 downstream from NOTCH1 (Sharma et al. 2006; Weng leukemias. We found fourfold higher NOTCH1 protein et al. 2006) and possibly NOTCH3. Furthermore, NOTCH1 levels in Cd3e+/+ tumors and strong nuclear staining (Fig. target genes were low in leukemic samples from mouse 3B; Supplemental Fig. S7A), indicating that mutations of the #241, in which the Notch1 gene was wild type and for PEST domain result in increased NOTCH1 protein. In- which the time of leukemia onset was the longest (183 d) creased nuclear NOTCH1 in Cd3e+/+ tumors was accom- within the SCL–LMO1 series. These results suggest that panied by higher expression of known NOTCH1 target NOTCH1 activity is higher in Cd3e+/+SCLtgLMO1tg tu- genes in SCLtgLMO1tg leukemias (Fig. 3C; Supplemental mors, concurring with higher NOTCH1 protein levels and Fig. S7B), Nrarp, Dtx1, Hes5, Hes6,andHey1. Hes1, Il7r, PEST domain mutations in the Notch1 gene. Gata3,andMyc did not differ between Cd3e+/+ and Cd3eÀ/À Strikingly, Notch1 mutations were found in DN4 cells leukemias. Finally, Notch3 and Ptcra showed higher ex- during the late preleukemic stage, but not in more primitive pression levels in Cd3eÀ/À leukemias. Since Hes1, Il7ra, thymocyte progenitors, except for sample #29191 (Fig. 3D). Myc,andPtcra are also controlled by NOTCH3 (Bellavia Furthermore, the same mutation was found in subsequent

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Notch1 interacts with SCL and LMO1 in T-ALL stages of thymocyte differentiation. However, mutations #26888 may be due to disrupted hierarchical organization were found at different positions in five different preleu- and abnormal differentiation in leukemia (Jordan 2009), kemic samples. Together, these observations further sup- or, alternatively, that DN1 cells can occasionally acquire port the hypothesis that the acquisition of Notch1 gain of Notch1 mutations. Again, all subpopulations have the same function confers clonal dominance. In sample #26897, we mutation as observed during the preleukemic phase, con- did not observe Notch1 mutations in the CD8 lineage, sistent with clonal dominance. Together, our observations possibly because of the limit of detection of the PCR re- indicate that Notch1 gain of function as well as an active action, or, alternatively, these cells are not functional pre-TCR collaborate with the SCL and LMO1 oncogenes. CD8+ cells, but represent more immature stages. These observations indicate that Notch1 mutations occur mostly Genetic interactions between Notch1 and SCL–LMO1 at the DN4 stage during the preleukemic phase, and concur with the absence of Notch1 mutations in Cd3- To determine whether Notch1 interacts genetically with deficient leukemias (Fig. 3A–D). At the time of overt the SCL and LMO1 oncogenes, we crossed SCLtgLMO1tg leukemia, however, we found Notch1 mutations in DN3 mice with mice overexpressing a constitutively active cells in two out of three samples. Since DN3 cells are intracellular NOTCH1, lacking the PEST and part of defined as CD25+CD44À and, furthermore, CD25 is a tar- the TAD domains (Notch1-ICDP), under the control of get of NOTCH (Deftos et al. 2000), it is possible that the the Lck promoter (Fowlkes and Robey 2002). Notch1tg or acquisition of Notch1 gain-of-function mutations may SCLtgLMO1tg mice developed acute leukemia with highly cause abnormal CD25 expression. The presence of Notch1 variable times of onset (Fig. 4A). The SCL or LMO1 trans- mutations in DN1 and DN2 subpopulations in sample gene did not induce leukemia separately, but significantly

Figure 4. Genetic interaction between SCL, LMO1, and a hyperactive Notch1 allele in leukemogenesis. (A) Survival analysis of SCLtgLMO1tg, Notch1tg, Notch1tgSCLtg, Notch1tgLMO1tg, and Notch1tg SCLtgLMO1tg mice. Results are presented as in Figure 2A ([*] P < 0.001 as compared with SCLtgLMO1tg mice). Cohorts of n mice were analyzed per genotype. (B) Preleukemic and leukemic phenotypes of Notch1tg and Notch1tgSCLtg LMO1tg mice. Representative flow cytometry analysis of thymocytes was performed at the indicated times as in Figure 2B. (C) Oligoclo- nal T-cell expansion detected by PCR analysis of Tcrb gene rearrangements in Notch1tg and Notch1tgSCLtgLMO1tg tumors. Genomic DNA was amplified by PCR with primers for specific variability segments of Tcrb. Shown is germline configuration, rearrangements in wild-type thymus, and seven independent Notch1tgSCLtgLMO1tg and five independent Notch1tg tumors. (D) Leukemic cells from SCLtgLMO1tg and Notch1tg SCLtgLMO1tg were transplanted through limiting dilution in Rag1À/À mice. Shown is the time to leukemia after transplantation of the indicated numbers of LSC equivalent. LIC frequency (Range LIC 6 SE) for the indicated genotype was calculated by applying Poisson statistics using the Limit Dilution Analysis software (Stem Cell Technologies). (E) Purified preleukemic thymocyte populations from 7-d-old SCLtgLMO1tg and Notch1tgSCLtgLMO1tg mice were transplanted into sublethally irradiated coisogenic Pep3B mice at a concentration of 3 3 104. Shown are the numbers of mice presenting T-ALL 4 wk post-transplantation over the total numbers of transplanted mice. (F) Penetrance and median time to leukemia for SCLtgLMO1tg, Notch1tg SCLtgLMO1tg, Cd3eÀ/À SCLtgLMO1tg, and Cd3eÀ/ÀNotch1tg SCLtgLMO1tg.

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Tremblay et al. accelerated leukemia onset in the presence of Notch1- of the leukemic clone. Strikingly, this time to leukemia ICDP, suggesting that these genes operate in parallel was comparable with that observed in Notch1tgSCLtg pathways. In all of these series, the time of onset remains LMO1tg mice (Fig. 4A). We therefore conclude that the highly variable. Strikingly, triple Notch1tgSCLtgLMO1tg 92-d difference between the median time to leukemia mice developed leukemia almost synchronously around of double SCLtgLMO1tg and triple Notch1tgSCLtgLMO1tg day 31 after birth. This shortened time of onset (P < 0.01) mice represents the time required for acquisition of Notch1 indicates synergistic interactions. Furthermore, this nar- mutations in a Cd3e+/+ background. row window of onset suggests that the introduction of To address the question of whether a constitutively Notch1-ICDP is sufficient to bypass the time required for active Notch1 gene confers leukemia-inducing potential clonal evolution, which is highly variable. to preleukemic SCL–LMO1-expressing DN or DP cells, we We next assessed the cellular distribution within the DN transplanted 3 3 104 purified DN1–DN2 and DN3–DN4 population of leukemic cells. In contrast to the SCL and cells and 3 3 105 DP cells from double SCLtgLMO1tg and LMO1 transgenes, Notch1-ICDP did not cause an expan- triple Notch1tgSCLtgLMO1tg mice into sublethally irradi- sion of the DN1/DN2 populations, but of the DN4 and ated hosts (Fig. 4E; data not shown). The results show that ISP8 (Supplemental Fig. S6C), even though the Notch1- purified thymocyte subsets from double SCLtgLMO1tg ICDP transgene may be expressed at earlier stages. Since mice failed to induce leukemia after 70 d, indicating that Notch1 mutations cosegregate genetically with Cd3 (Fig. these two oncogenes are not sufficient for cell transforma- 3A), and Notch1 mutations are found in DN4 cells and tion. This contrasts with the capacity of leukemic DN3/4 later stages, we surmise that the proliferative signal from cells to initiate leukemia in transplanted hosts, possibly the pre-TCR increases the likelihood of accumulating mu- due to acquired Notch1 mutations. Indeed, purified DN1– tations that include the Notch1 locus. In leukemic thymi, DN2 and DN3–DN4 cells from triple Notch1tgSCLtg coexpression of the SCL–LMO1 transgenes had expanded LMO1tg mice induced T-ALL in secondary recipients with the numbers of DN1 and DN2 cells compared with leu- high efficiency, indicating that Notch1 gain of function kemias arising in Notch1tg mice, whereas the other pop- confers leukemogenicity to SCL–LMO1-expressing DN ulations did not differ significantly between the two types of thymocytes. In contrast, the DP population was much leukemias. Notably, the heterogenous distribution within less susceptible, and only one of six recipient mice de- each DN or DP subset was markedly reduced in triple- veloped leukemia. Moreover, the transformation of DN1– transgenic mice compared with Notch1tg or SCLtgLMO1tg DN2 cells by the three oncogenes indicates that pre-TCR mice (Supplemental Figs. S4B-S6C). In addition, Cd3eÀ/À is not essential for leukemogenesis. Accordingly, we leukemias differ most from triple-transgenic mice; i.e., the observed that the Notch1 transgene allows for a bypass numbers of DN1 and DN4 cells were lowest and the of pre-TCR signaling deficiency to induce T-ALL with full number of DP was highest, consistent with the absence of penetrance and shortened latency on a Cd3-deficient Notch1 mutations in these leukemias. However, the ISP8 background (Fig. 4F). Finally, all preleukemic triple- population was in the same range, indicating that the SCL– transgenic DN subsets induced T-ALL within 25 d, which LMO1 oncogenes can expand this population in the absence corresponds to the time required for clonal expansion of of Notch1 gain-of-function mutation. Overall, the total fully transformed LSCs. Together, these experiments in- numbers of cells within the various DN subsets in leuke- dicate that the three oncogenes—Notch1, SCL,and mias arising in triple Notch1tgSCLtgLMO1tg mice was not LMO1—are sufficient to convert susceptible thymocyte higher than those arising in SCLtgLMO1tg or Notch1tg mice precursors into LICs without requiring pre-TCR signaling. (Supplemental Fig. S6C). We next verified the clonality of Our observations concur to support the conclusions leukemias arising in Notch1tgSCLtgLMO1tg mice and traced that the variable times of leukemia onset in Cd3e+/+ the presence of numerous clones (Fig. 4C), contrasting with SCLtgLMO1tg mice represents the time required for the predominance of one or a few clones in SCLtgLMO1tg accumulation of Notch1 mutations (Fig. 5A). Accord- leukemias (Supplemental Figs. S3, S4B). The presence of ingly, the presence of all three oncogenes at birth is multiple clones in leukemias from Notch1tgSCLtgLMO1tg sufficient for full transformation of primary thymocyte mice suggests that T-ALL occurred in the absence of precursors. The time to leukemia therefore represents the obvious clonal dominance. time required for clonal expansion (Fig. 5B). To determine whether the shortened time to leukemia in triple Notch1tgSCLtgLMO1tg compared with SCLtgLMO1tg Discussion mice might be due to increased LIC frequencies, we transplanted Rag1À/À mice with limiting dilutions of cells from In the present study, we provide genetic evidence that these leukemias (Fig. 4D). We found that LIC frequency Notch1 and the pre-TCR collaborate with the SCL–LMO1 was not increased by the Notch1 transgene. Furthermore, oncogenes in T-ALL. this limiting dilution analysis indicates that the time to The pre-TCR and Notch1 as collaborating events leukemia for one leukemic stem cell (LSC) equivalent is in leukemogenesis 31dforNotch1tgSCLtgLMO1tg leukemias and 36 d for SCLtgLMO1tg leukemias. These similarities concur with Primary oncogenic events such as those triggered by chro- the presence of spontaneously occurring Notch1 mutations mosomal translocations are generally insufficient by them- in SCLtgLMO1tg leukemias and with the determining im- selves to cause leukemias, and require secondary coopera- portance of these gain-of-function mutations on the biology tive mutations to fully transform cells. These collaborating

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Notch1 interacts with SCL and LMO1 in T-ALL

present study, we provide evidence that the aggressiveness of pre-TCR-proficient SCL–LMO1 T-ALL is associated with Notch1 mutations. Activating Notch1 mutations are a fre- quent occurrence in T-ALL and have been shown to coincide with the appearance of pre-LSCs in SCLtgLMO1tg mice (Lin et al. 2006). The absence of Notch1 gain-of- function mutations in Cd3eÀ/À leukemias suggest that either Notch1 mutations do not provide a selective advantage to pre-TCR-deficient cells, or these cells are not permissive to Notch1 mutations. Here we provide several lines of evidence in support of the second hypothesis. First, we show that SCL–LMO1 expands ETP-DN1 cells and subsequent DN stages of thymocyte differentiation but inhibits the DN4–DP transition, thus favoring the accumulation of cells at the highly proliferative DN4 stage and, consequently, the appearance of additional mutations. Second, we found that activating Notch1 mutations cosegregate with Cd3e in leukemogenesis. Third, in Cd3e-pro- Figure 5. Model of progression to leukemia. (A) At the time of ficient cells, Notch1 mutations are found in DN4 cells and weaning, thymic expression of the SCL–LMO1 oncogenes in- later maturational stages, but not in DN1/2 and rarely in duces an expansion of the ETP/DN1 to DN4 population. A period of 92 d is necessary for acquisition of Notch1 gain-of-function DN3 cells. Nonetheless, a hyperactive Notch1 allele by- mutations. The clonal expansion of the leukemic population passes the requirement for pre-TCR function in SCL–LMO1- takes another 31 d before overt leukemia. (B) The presence of all induced leukemias. In summary, our observations indicate three oncogenes (Notch1, SCL,andLMO1) at birth is sufficient that the pre-TCR provides a permissive context for the ap- for full transformation of primary thymocyte precursors. The pearance of mutations that include gain-of-function muta- time to leukemia therefore represents the time required for clonal tions of the Notch1 gene in primary thymocyte precursors expansion. expanded by the SCL–LMO1 oncogenes. Finally, our observation that, during the preleukemic phase, this hy- events may be different, depending on the initiating cyto- peractive Notch1 allele confers leukemogenic potential to genetic event and the target cell of transformation. Hence, SCLtgLMO1tg DN1–DN2 subsets that are pre-TCR-indepen- T-ALL with MLL rearrangements exhibit high Flt3 lev- dent indicates that Notch1 can provide a selective advantage els (Armstrong et al. 2002; Brown et al. 2005), whereas in the absence of the pre-TCR (Campese et al. 2006). NOTCH1-activating mutations were found in most mo- A recent study indicates that LMO2 but not Notch3 lecular subgroups of T-ALL (Weng et al. 2004; Grabher et al. favors the self-renewal of DN3 thymocytes (McCormack 2006). et al. 2010), leaving open the question of the role of Notch1 Previous work indicates that pre-TCR or TCR signaling gain-of-function mutations in T-ALL. Our observations (Chervinsky et al. 2001; Fasseu et al. 2007) accelerates SCL- indicate that activating Notch1 mutations are essential dependent leukemias in transgenic mouse models, and that for cell transformation induced by the SCL and LMO1 on- SCL+ T-ALL constitutes a poor prognostic group in child- cogenes. Previous work places the Myc oncogene down- hood leukemias (Ferrando et al. 2002), associated with stream from NOTCH1 (Sharma et al. 2006; Weng et al. elevated expression of genes of the TCR pathway. The re- 2006; Li et al. 2008). As a corollary, Notch1 and Myc mu- lative importance of the pre-TCR or the TCR, however, tations are mutually exclusive in large-scale insertional remains debatable. Here, we show that abrogation of an- mutagenesis screens (Kool and Berns 2009). Furthermore, tigen presentation in the AbbÀ/Àb2mÀ/À mice did not SCL-induced leukemias are also accelerated by loss of delay SCL–LMO1-induced leukemias, indicating that, Trp53 (Curtis et al. 1997), and Notch1 has been shown to when the pre-TCR is active, the contribution of antigen- suppress p53 in lymphomagenesis (Beverly et al. 2005), dependent TCR signaling to leukemogenesis is minimal. In consistent with the observations that activating Notch1 contrast, CD3 signaling is an important determinant, mutations are not found in p53-deficient lymphomas (Uren consistent with the report that all SCL–LMO1-induced et al. 2008). It will be interesting to assess whether p53 T-ALLinSCIDmicehaveanin-framerearrangedTCRb deficiency or c-Myc acts downstream from or in parallel and express the pTa (Chervinsky et al. 2001), suggesting to Notch1 in SCL–LMO1/2-induced leukemias. Although a functional importance for the pre-TCR. Nonetheless, Notch1 mutations occur at a high frequency of ;80% in when the pre-TCR is inactive, as in pTa-deficient mice in Cd3e+/+SCLtgLMO1tg mice (Lin et al. 2006; this study), cell which TCRa/b lymphocytes are still produced, albeit at transformation can also involve alternative pathways, such reduced levels, it is possible that the TCRa/b or TCRg/d as the Wnt/b-catenin signaling pathway (Guo et al. 2007). may provide an alternative collaborative signal for the SCL–LMO1 oncogenes, as reported (Fasseu et al. 2007). Target cell of transformation Despite the recognition that pre-TCR signaling is important in T-ALL, the precise contribution of the pre-TCR The nature of the target cells of transformation by onco- to leukemogenesis remains to be documented. In the proteins remains an open question. The DP population is

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Tremblay et al. transient in the thymus, and most DP thymocytes are initiating potential, even though leukemic DN3–DN4 eliminated by apoptosis. Thus, even if oncogene activation cells have the highest potential in our model. During the can occur in these cells, the probability of survival and preleukemic phase, Notch1 mutations occur in DN4 subsequent clonal evolution is limited. Our genetic ap- thymocytes. At time of overt leukemia, however, Notch1 proach, combined with transplantation of purified thymo- mutations were identified in leukemic cells with a more cyte progenitors during the preleukemic phase (i.e., 1 wk primitive phenotype, which could be explained by either a after birth), indicates that the collaboration between the developmental flexibility (Jordan 2009) or oncogene-medi- three oncogenes (SCL, LMO1,andNotch1) is sufficient to ated surface marker misexpression. Indeed, in Notch1tg transform susceptible target cells; i.e., DN1–DN4 thymo- thymocytes, we observed an increase in Cd25 expression cyte progenitors. In principle, all hematopoietic progeni- (Deftos et al. 2000; Fowlkes and Robey 2002; data not tors that can give rise to thymocytes could be targeted by shown) that could confer a DN3 surface phenotype to cells the Notch1 oncogene. Indeed, retroviral delivery of a con- that are actually at the DN4 stage. Together, our results stutively active NOTCH1 gene in hematopoietic stem show that different subpopulations of leukemic cells have cells (HSCs) has been shown to exclusively induce T-ALL leukemia-initiating potential, suggesting that leukemic (Pear et al. 1996). In contrast, our observations indicate cells in T-ALL do not follow an orderly developmental that DP cells are less susceptible to cell transformation by structure. these oncogenes. Since the DP stage represents a critical checkpoint in the thymus, it is possible that only DP cells Significance of the time to leukemia that have successfully undergone selection are prone to oncogene transformation, whereas cells that are fated to be In our transgenic model, SCL and LMO1 gains of functions eliminated may require additional collaborating events. precede the acquisition of Notch1 mutations (Fig. 5; Lin Since SCL–LMO1/2 leukemias are mostly DP, these et al. 2006), although the sequence of events may also be observations are consistent with the view that the surface inverted, as reported in some cases of childhood T-ALL phenotype of the bulk of leukemic cells may not be in- (Eguchi-Ishimae et al. 2008; Hong et al. 2008). It has been dicative of the cell of origin of leukemia. This is further proposed that acquiring the appropriate numbers of com- supported by our genetic approach that ectopic expres- plementary genetic hits may be more important than the sion of the SCL–LMO1 oncogenes in the thymus of Rag1- sequence of events in cell transformation, even though or Cd3e-deficient mice induces tumors that ‘‘illegiti- these genetic alterations may occur according to a preferred mately’’ express the CD4 and/or CD8 markers (DP or sequence (Fearon and Vogelstein 1990). In the present ISP8), even though these thymocytes should be arrested study, we provide several lines of evidence supporting the at the DN3 stage due to pre-TCR deficiency. hypothesis that Notch1 gain-of-function mutations confer Similar to Notch1,ectopicLMO2 expression in bone clonal dominance to SCL–LMO1 transformed cells. First, marrow HSCs by retroviral gene transfer (our unpublished the clonality of leukemic cells as assessed by the Tcrb re- results) or by LTR-mediated activation of the endogenous arrangement indicates the emergence of a single clone. gene (for review, see Hoang 2010) also gives rise to T-ALL. Second, seven of eight Cd3e-proficient leukemias exhibit Thus, bone marrow-derived HSCs and all DN thymic Notch1-activating mutations. Third, in the absence of populations are susceptible to transformation by NOTCH1, Cd3e and activating Notch1 mutations, tumor cells have SCL, and LMO1. Nonetheless, in the double SCL–LMO1 decreased leukemia-initiating potential. transgenic mouse model, Notch1 mutations accumulate at At the time of overt leukemia, tumor cells, in principle, the DN4 stage, possibly due to intensive cell proliferation. have acquired the minimum numbers of transforming Since Notch1 gain of function confers leukemia-initiating events. Therefore, the time taken by a single LSC to in- potential to SCL–LMO1 transgenic thymocytes, it is quite duce leukemia in secondary congenic hosts represents possible that SCL–LMO1 favors the accumulation of DN4 the time required for clonal expansion. We found that the cellsbyexpandingtheDN1population,butalsobypre- time to leukemia for a single LSC is 36 d, consistent with venting the DN4–DP transition, thereby facilitating the results published for another genetic model of T-ALL in acquisition of complementing Notch1 mutations. mice caused by PTEN deficiency (Guo et al. 2008). In- LSCs have been studied extensively in acute myeloid terestingly, leukemia develops with a latency of 31 d in leukemia (AML), where they are found in primitive pop- Notch1tgSCLtgLMO1tg mice. Furthermore, transplanta- ulations (Lapidot et al. 1994). In contrast, the identification tion of 7-d-old preleukemic triple-transgenic thymocytes of LSCs in ALL is still controversial (Jordan 2009). A recent gives rise to leukemia in recipient mice after 3 wk, thus study in human B-ALL showed that most populations of recapitulating the above time of tumor onset. This latency leukemic blasts were able to reconstitute the tumor corresponds to the production of ;1010 cells from a single phenotype (le Viseur et al. 2008). Transformed leukemic LSC, assuming a 24-h division time, comparable with the cells could follow a disorganized differentiation pattern tumor mass collected from the thymi and lymph nodes of associated with an illegitimate expression of surface mark- these mice. We therefore surmise that pre-LSCs might ers resulting from oncogenic protein misexpression, such appear during the late prenatal period, as reported for as NOTCH1/3 (Michie et al. 2007). Despite the fact that ALL in monochorionic twins (Eguchi-Ishimae et al. 2008; preleukemic DP cells rarely initiate leukemias in trans- Hong et al. 2008). Furthermore, our observations suggest planted hosts, we show here that both leukemic DN and that SCL, LMO1,andNotch1 gains of functions, together DP populations in SCL–LMO1 leukemias have leukemia- with an active pre-TCR, might be sufficient to transform

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Notch1 interacts with SCL and LMO1 in T-ALL thymocytes. As a corollary, the 92-d difference in median with FITC-conjugated donkey anti-rabbit antibody (Jackson survival times between the double SCLtgLMO1tg model and ImmunoResearch Laboratories) for 45 min at room temperature. the triple Notch1tgSCLtgLMO1tg model likely represents The coverslips were then washed three times, mounted in Vecta- the time required for the occurrence of Notch1 mutations shield with DAPI (Vector Laboratories, Inc.), and sealed. Fluores- in appropriate target cells. Our observations are consistent cence was analyzed on a confocal microscope LSM 510 META (Zeiss) with a 1003 objective, further magnified by a zoom of 4. with the view that SCL–LMO1 oncogenes expand the DN populations, increasing the likelihood of acquisition of Notch1 mutations in DN4 thymocytes, thereby conferring FACS analysis clonal dominance to transformed cells. Single-cell suspensions were prepared from thymi or thymoma of mice at the indicated ages. Flow cytometry analysis and cell sorting were done as described previously (Herblot et al. 2000) Materials and methods using Pharmingen antibodies (BD Biosciences) against CD44 (IM7), CD25 (PC61.5), CD4 (RM4-4), CD8 (53-6.7), Thy1.2 (30- Mouse models H12), TCRb (H57-597), and CD3e (145-2C11). Bone marrow KSL We used the previously described pSil-TSCL (SCLtg)(Aplanetal. population was stained as described previously (Lacombe et al. 1997) and Lck-LMO1 (LMO1tg) (McGuire et al. 1992) transgenic 2009). ETPs (LinÀSca1+CD25À) were analyzed by staining thymo- mouse lines (Herblot et al. 2000). Both mouse lines were back- cytes with c-KIT (eBioscience, 2B8), SCA1 (BD, E13-161.7), and crossed onto a C57BL6/J background for >12 generations. All CD25 antibodies and excluding Lin+ cells stained with biotiny- mouse strains were on a C57BL6/J background: Rag1À/À (Jackson lated antibodies against B220 (RA3-6B2), TER119 (eBioscience, Laboratory), AbbÀ/ÀB2mÀ/À (Taconic), Lck-NotchIC9 (Notch1tg) TER119), CD11b (M1/70), GR1 (RB6-8C5), CD8a (53-6.7), CD3e (Fowlkes and Robey 2002) (NIAID/Taconic Repository, Bethesda), (145-2C11), NK1.1 (PK136), TCRb (H57-597), TCRgd (GL3), and and Cd3eÀ/À kindly given by P. Hugo (Malissen et al. 1995). Mice CD11c (HL3). Dead cells were excluded by propidium iodide cohorts were generated by cross-breeding, and their genotypes staining. FACS analysis was performed on an LSRII cytometer, were verified by PCR. Transplantation assays were performed by and cell sorting was performed on FACSAria (BD Biosciences). injection of leukemic thymoma cells into unirradiated Rag1À/À mice or purified subpopulations of preleukemic cells into isogenic Acknowledgments Pep3B mice. All animals were maintained in pathogen-free con- ditions according to institutional animal care and use guidelines. We thank Danie`le Gagne´ (IRIC) for her assistance with cell sorting, Kaplan-Meier survival and statistical analysis was performed and Veronique Litalien for mice handling. This work was funded using GraphPad Prism 4.0 software (GraphPad Software, Inc.). by grants from the Canadian Cancer Society Research Institute (T.H.) and the Canada Research Chair program (T.H.). The Quebec Leukemia Cell Bank is supported in part by funds from the Fonds Notch1 sequencing de Recherche en Sante´ du Que´bec (FRSQ) and by the Cole Amplification of Notch1 exons 26, 27, and 34 from cDNA of Foundation (J.H.). The flow cytometry service was supported by tumor cells was performed by PCR. Amplification products a Multiuser grant from the Canadian Institute for Health Research were sequenced in both directions. Primer sequences are shown (CIHR), and the infrastructure was supported in part by an FRSQ in Supplemental Table S1. Residues are numbered according group grant (IRIC). M.T. received doctoral awards from CIHR and to their location in Notch1 sequence (Ensembl sequence the Cole Foundation, S.H. received a post-doctoral fellowship from ENSMUSG00000026923). the Leukemia Research Fund of Canada, and C.S.T. received a post-doctoral Research Fellowship of the Terry Fox Foundation Award (#700153). Clonality analysis

Genomic DNA was obtained from digestion of leukemic cell References extracts with proteinase K, followed by phenol/chloroform extraction. Tcrb rearrangements were determined by PCR ampli- Aifantis I, Raetz E, Buonamici S. 2008. Molecular pathogenesis fication (Aplan et al. 1997). of T-cell leukaemia and lymphoma. Nat Rev Immunol 8: 380–390. Aplan PD, Jones CA, Chervinsky DS, Zhao X, Ellsworth M, Wu RNA analysis C, McGuire EA, Gross KW. 1997. An scl gene product Total RNA was prepared from 10,000 sorted cells as described lacking the transactivation domain induces bony abnormal- previously (Herblot et al. 2000) using RNeasy extraction kit ities and cooperates with LMO1 to generate T-cell malig- (Qiagen). Primer sequences used are shown in Supplemental nancies in transgenic mice. EMBO J 16: 2408–2419. Table S1. Real-time quantitative PCR was done with SYBR Armstrong SA, Staunton JE, Silverman LB, Pieters R, denBoer Green detection kits (Qiagen) on a Stratagene Mx3000 apparatus ML, Minden, MD, Sallan, SE, Lander, ES, Golub, TR, (Stratagene). Korsmeyer, SJ. 2002. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 30: 41–47. Microscopy Armstrong F, Brunet de la Grange P, Gerby B, Rouyez MC, Calvo Thymoma cells were rinsed twice in PBS (pH 7.4), fixed in J, Fontenay M, Boissel N, Dombret H, Baruchel A, Landman- 2% paraformaldehyde for 10 min at room temperature, washed, Parker J, et al. 2009. NOTCH is a key regulator of human blocked, and permeabilized with 10% FBS and 0.1% NP40 in PBS T-cell acute leukemia initiating cell activity. Blood 113: (pH 7.4). Cells were then incubated with rabbit anti-NOTCH1 1730–1740. antibody (C20R, sc-6014R, Santa Cruz Biotechnologies) in block- Begley CG, Aplan PD, Davey MP, Nakahara K, Tchorz K, ing buffer for 2 h. After incubation with primary antibodies, the Kurtzberg J, Hershfield MS, Haynes BF, Cohen DI, cells were washed three times in 13 PBS (pH 7.4) and incubated Waldmann TA, et al. 1989. Chromosomal translocation in a

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Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes

Mathieu Tremblay, Cédric S. Tremblay, Sabine Herblot, et al.

Genes Dev. 2010, 24: Access the most recent version at doi:10.1101/gad.1897910

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