REVIEW Notch and Wnt Signaling in T-Lymphocyte Development And

Notch and Wnt signaling in T-lymphocyte development and acute lymphoblastic leukemia

F Weerkamp, JJM van Dongen and FJT Staal

Department of Immunology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands

Many acute lymphoblastic leukemias can be considered Wnt proteins, these are factors of the Tcf/Lef family, in as malignant counterparts of cells in the various stages thymocytes mainly Tcf-1. of normal lymphoid development in bone marrow and thymus. T-cell development in the thymus is an ordered and tightly Both Notch and Wnt proteins were first identified in controlled process. Two evolutionary conserved signaling Drosophila and named after their effect in the development pathways, which were first discovered in Drosophila, of the Drosophila wing: heterozygous mutation of Notch control the earliest steps of T-cell development. These are resulted in a nicked wing phenotype,1 whereas the ‘wingless’ the Notch and Wnt-signaling routes, which both are deregu- mutation of Wnt dramatically shortened the wing.2 Attention lated in several types of leukemias. In this review we discuss was attracted to these genes in the mammalian system when it both pathways, with respect to their signaling mechanisms, was discovered that both function as proto-oncogenes. Nusse functions during T-cell development and their roles in devel- 3 opment of leukemias, especially T-cell acute lymphoblastic and Varmus discovered the Wnt gene (then termed Int1)asan leukemia. integration site for mouse mammary-tumor viruses. The first Leukemia (2006) 20, 1197–1205. doi:10.1038/sj.leu.2404255; mammalian homologue of the Drosophila Notch gene was published online 11 May 2006 identified as TAN1, the fusion partner of the T-cell receptor beta Keywords: Notch; Wnt; T-cell development (TCRB) gene in t(7;9) causing T-cell acute lymphoblastic leukemia (T-ALL).4 Later it was found that both Notch and Wnt signaling play Introduction critical roles in normal T-cell development. Both cascades are thought to mediate different processes in the thymus: It is becoming increasingly clear that most cancers arise Notch in differentiation and Wnt in proliferation, although from cancer stem cells and follow differentiation pathways we will show in this review that this is not as clear-cut as that closely resemble the developmental and ontogenetic assumed. Despite differences in function and their very different pathways of normal cells and tissues. This is in particular molecular mechanisms, many parallels exist for both pathways. the case for antigen-specific cells of the immune system, the Absence of either causes a block at the earliest stages of lymphocytes. There is no other developmental system that T-cell development, but both are thought to be important for has been described to such detail as the differentiation of later stages as well. Interestingly, the two signaling pathways B lymphocytes in the bone marrow (BM) and of T lymphocytes also play a role in hematopoietic stem cell (HSC) self-renewal. in the thymus. In order to fully comprehend the malignant Moreover, dysregulated signaling of both cascades is involved in processes underlying lymphoid leukemias, the malignant leukemogenesis. counterpart of lymphocytes, thorough knowledge of normal differentiation pathways is a prerequisite. T cells develop in the thymus, from BM-derived multipotent Overview of T-cell development in the thymus progenitors. The ‘thymus-seeding’ cells encounter molecules that initiate and guide their development: adhesion molecules, T-cell development takes place in the thymus, a primary cytokines, chemokines and morphogens largely produced by lymphoid organ that provides a unique microenvironment for the thymic stroma. These molecules initiate signaling path- the development of hematopoietic progenitors into mature ways that lead to the activation of transcription factors that T cells.5 In adult mice and humans, these precursor cells are change the transcriptional program of a cell. Two exogenously derived from the BM, whereas during fetal development the activated signaling pathways that are crucial for thymocyte liver is the principal source. The exact nature of the progenitors development and have attracted much attention recently is unclear, but some level of multipotency is now commonly are the Notch and the Wnt-signaling cascades. Both act as accepted.6,7 Development into a mature T cell comprises the molecular switches: the executing transcription factor that is ordered rearrangement of the different TCR genes and positive normally bound to the DNA as a transcriptional repressor is and negative selection events that finally lead to the expression converted into an activator. For the Notch pathway, set in of mature TCRab and TCRgd molecules recognizing non-self motion by ligands of the Delta and Jagged family, this peptides in context of self-major histocompatibility complex transcription factor is CSL (see section on ‘Notch signaling’ (MHC) or equivalent molecules. These processes take place below). For the Wnt pathway, which is activated by secreted during a series of discrete phenotypic stages that can be recognized by expression of several key membrane molecules, Correspondence: Dr FJT Staal, Department of Immunology, Erasmus most notably CD4 and CD8 (Figure 1a). In both man and mouse, MC University Medical Center Rotterdam, Dr Molewaterplein 50, thymocytes are successively CD4ÀCD8À (double negative 3015 GE Rotterdam, The Netherlands. þ À E-mail: [email protected] (DN)), CD4 CD3 (human immature single positive (ISP)) or þ À þ þ Received 3 March 2006; revised 3 April 2006; accepted 6 April 2006; CD8 CD3 (mouse ISP), CD4 CD8 (double positive (DP)) published online 11 May 2006 and finally CD4 þ CD3 þ or CD8 þ CD3 þ (single positive Notch and Wnt signaling in T-cell development and T-ALL F Weerkamp et al 1198

Figure 1 Schematic representation of three thymic lobules. (a) The different thymocyte subsets and their migration through the anatomic niches. The cells that enter the thymus are CD4ÀCD8À double negative (DN). The thymocytes subsequently develop into immature single positive (ISP), double positive (DP) and single positive (SP) stages. Finally, mature naive T cells leave the thymus into the blood. (b) The primary events during ab T-cell development. The earliest DN cells in the thymus massively proliferate. In the DN3 stage, rearrangements of the TCRB genes start. Production of a successful TCRb chain leads to membrane expression of the pre-TCR in the ISP cells, after which proliferation and initiation of TCRA rearrangements occurs. During the DP stage, positive selection (for a functional TCR) and negative selection (against self-reactivity) takes place. Surviving cells become SP. (c) The stromal compartments of the thymus. Each compartment contains specialized epithelial cells (TECs), which provide Wnt proteins, Notch ligands, and other growth and differentiation factors for developing thymocytes. The earliest thymocytes enter the thymus at the cortico-medullary junction and migrate to the subcapsular zone. As they mature, they pass through the cortex and the medulla.

(SP)).8,9 In the mouse, the DN subset can be further subdivided Notch signaling in the thymus into four stages: CD44 þ CD25À (DN1), CD44 þ CD25 þ (DN2), CD44ÀCD25 þ (DN3) and CD44ÀCD25À (DN4).10 In the The Notch signal transduction pathway, depicted in Figure 2, is human thymus, CD34 þ CD1aÀ cells correspond to murine an evolutionary conserved mechanism that regulates cell DN1 and 2 thymocytes and CD34 þ CD1a þ cells are homo- fate determination during developmental processes.1 The Notch logous to DN3 cells 9,11 (Figure 1a). The human DN4 family of transmembrane receptors consists of four members, population is hard to distinguish from ISP cells that express named Notch1 to 4. Signaling is initiated when the large low level CD3. extracellular domain of the Notch receptor binds a membrane- As the numbers of progenitors that enter the thymus are bound ligand on a neighboring cell. The five Notch ligands in limited, an enormous expansion takes place during the DN1 mammals are Delta1, 3 and 4 and Jagged1 and 2. The and DN2 stages of T-cell development12 (Figure 1b). In the glycosylation status of the Notch receptor can be modified by DN3 stage, cells stop proliferating and start rearranging the Fringe proteins, thereby influencing ligand binding.17 TCRB loci, resulting in a complete TCRb chain during Interaction of Notch with a ligand can induce proteolytic the DN4/ISP stage.11 When the thymocytes have developed cleavage of Notch, successively by metalloprotease and into ISP cells, the functionality of the TCRb chain is tested g-secretase activities,18,19 releasing the intracellular part of the by expression on the cell surface together with a pre-TCR protein (intracellular (IC-) Notch). IC-Notch then translocates alpha (pTa) chain, a process called b-selection.13 Signals to the nucleus and binds to the nuclear transcription factor emanating from this pre-TCR complex induce the cells first to CSL (CBF1¼ c-promoter binding factor in humans, suppressor proliferate and subsequently to initiate TCRA gene rearrange- of hairless in Drosphila, Lag-1 in Caenorhabditis elegans; also ments. The thymocytes now become DP cells. Productive called RBP-k in mouse).20 Binding of IC-Notch to CSL induces TCRA rearrangements lead to the expression of a TCRab the dislocation of co-repressors (coR) such as Mint and Nrarp, complex on the cell surface, which is then tested for the and recruitment of co-activators (coA), such as Mastermind recognition of self-MHC molecules (positive selection) and (Maml), consequently stimulating the transcription of Notch absence of reactivity against self-antigens (negative selection).14 target genes20 (Figure 2). It has been estimated that 95% of developing thymocytes die The ligands that induce Notch signaling are present on the by apoptosis.15 The surviving cells become SP thymocytes membrane of stromal cells such as TECs. High levels of Notch that mature into either CD4 þ T helper cells or CD8 þ cytotoxic ligands Jagged1 and 2 and Delta1 and 4 are expressed in the T cells, and migrate to the periphery as naı¨ve recirculating fetal and adult murine thymus.21–24 Expression of Notch ligands T lymphocytes (Figure 1b). has been reported in murine and human BM stroma (Jagged1 The stages of T-cell development take place in different 25,26), as well as in murine fetal liver (mainly Delta1 and 423). stromal niches in the thymus and are guided by factors produced Notch receptors are expressed by hematopoietic progenitor cells by thymic epithelial cells (TECs) (Figure 1c). The proliferation, in the BM and fetal liver22,27–29 and by DN thymocytes differentiation and TCR rearrangement steps are tightly regulated (predominantly Notch1 and Notch324,29,30). by a number of molecular pathways (recently reviewed by When Notch-expressing hematopoietic progenitor cells enter Rothenberg and Taghon16) of which Notch and Wnt signaling the thymic microenvironment and interact with Notch ligands are perhaps the most conspicious. on the thymic epithelium, they immediately start expressing

Leukemia Notch and Wnt signaling in T-cell development and T-ALL F Weerkamp et al 1199 expression of Jagged in stroma cell lines,39 although Jagged, in another study, did inhibit development into B cells.41 Further- more, ab-lineage T-cell development was normal in Jagged2- deficient mice.43 Signals induced by Jagged may be involved in promoting NK cell or TCRgd þ cell differentiation.41,43,44 To further complicate matters, stable induction of a T-cell fate by Delta is dependent on contact time45 and density of ligands, with lower doses of Delta also promoting B-cell development.46 Furthermore, the interaction of Notch with the different ligands can be modulated by Fringe proteins.17 Overexpression in the thymus of Lunatic Fringe, which is thought to inhibit Notch activation by Jagged and facilitate interaction with Delta,47 blocked T-cell commitment.48 The downstream mechanisms by which a Notch signal is translated into a T-cell program are still largely unclear. The best known Notch target genes encode Hairy-Enhancer of Split (Hes)1 and Hes5 and Hes-related repressor protein (Herp), both basic-helix-loop-helix proteins that function as transcriptional repressors.49,50 Indeed overexpression of Hes1 and Hes5 in the BM partly inhibits B-cell development.51 But although Figure 2 The Notch-signaling pathway. The Notch receptor consists proliferation of early thymocytes is severely affected by Hes1 of a large extracellular part containing multiple EGF-like repeats and deficiency,52,53 thymocytes still develop in these mice. Also in an intracellular part that mediates Notch signal transduction. Binding human progenitors, transfection of Hes1 did not promote of the extracellular part to ligands of the Delta and Jagged families can differentiation into the T-cell lineage.54 Hes can therefore not be modulated by Fringe proteins. Interaction of Notch with a ligand induces proteolytic cleavage of Notch, successively by metallopro- be the sole target of Notch signaling in the thymus. By providing tease and g-secretase activities, releasing the intracellular part of the Notch signals to human progenitors in vitro, we could confirm protein (intracellular (IC-) Notch). IC-Notch then translocates to the HES1, NRARP and NOTCH1 itself as being genuine Notch nucleus and binds to the nuclear transcription factor CSL. Deltex1 and target genes, but not DTX1 (the gene for Deltex-1) and PTCRA Numb are cytoplasmic regulators of Notch signaling. Binding of IC- (the gene for pTa).56 Furthermore, we identified previously Notch to CSL induces the dislocation of co-repressors (coR) and unknown Notch targets, which are currently being tested recruitment of co-activators (coA) such as Mastermind, and stimulates the transcription of Notch target genes. The best known Notch-target functionally. We did not find any clues that Notch signaling gene is HES1 (Hairy-Enhancer of Split 1). can directly induce a T-cell fate, consistent with experiments of Taghon et al.55 in which murine fetal liver progenitors were cultured on stromal cell line OP9 transfected with Delta1. Notch target genes.22 These Notch signals are essential for This study showed that GATA3, TCF1 and PTCRA began to be the induction of a T-cell fate in progenitor cells. This was first expressed after three days of culture on OP9-Delta1, whereas demonstrated in mice in which the NOTCH1 gene was high HES1 transcription was detected already after one day. conditionally deleted, resulting in a complete block in T-cell These findings indicate that either Notch signaling induces development at the DN1 stage31 and the emergence of ectopic T-cell genes in already more differentiated thymocytes, or Notch B-cell development in the thymus.32 These effects are likely signaling stimulates the expression of other transcription factors, mediated by Notch1 signaling through CSL, as CSL-deficient which in turn activate or repress lineage differentiation genes. mice display a phenotype similar to Notch1-deficient mice.33 In all, it is clear that although Notch is crucial for T-cell Several reports suggest alternative Notch-signaling pathways development, it is not the sole signal responsible for inducing that are CSL-independent and may involve Deltex as an effector T-cell lineage commitment. We have recently elaborated on this protein,34 but their importance for T-cell development is not point elsewhere.56 yet known. While inhibition of Notch signaling blocks T-cell The Notch pathway is active in and necessary for T-cell development, BM reconstitution of mice with cells transduced development in the earliest thymocyte populations,57,58 but has with IC-Notch instructed a T-cell fate in BM progenitors and also been implicated in later stages of T-cell development, inhibited B-cell development.35 Similar results were obtained mostly in the DN to DP transition.59–63 Furthermore, mice when mice were transplanted with human hematopoietic in which Notch1 was deleted after the T/B-fate choice progenitors overexpressing IC-Notch1 or IC-Notch4.27,36 (Lck-cre Â Notch1lox/lox) show a complex phenotype which The expression of the different Notch ligands and their suggest that Notch1 contributes to both V to DJ rearrangement receptors in a stage- and tissue-specific manner raises of the TCRB locus as well as to the physical elimination of cells the possibility of a unique function for each ligand.24 However, that fail b-selection.64 the mechanisms by which specific ligands induce different There are conflicting data on a function of Notch signaling in cellular fates remain unclear. Transplantation of mice with promoting ab-lineage T-cell development at the expense of gd progenitors overexpressing Delta1 or 4 phenocopied the effect T cells64–66 and in the selection between CD4 and CD8 SP of overexpression of IC-Notch: DP cells emerged in the BM.37,38 thymocytes.61,62,67–69 In light of the focus of this review we will In vitro, this phenomenon can be mimicked using BM-derived not elaborate on these topics. stromal cell lines (e.g. S17 or OP9) which otherwise promote myeloid and B-cell development of hematopoietic progenitor cells. Forced expression of Delta1 in these stromal cell lines Wnt signaling in the thymus induces murine and human progenitor cells to develop into T cells, in some studies up to DP and SP stages.39–42 The striking Wnt genes encode a large family of secreted glycoproteins that T-cell generating effect of Delta could not be reproduced by regulate cell-to-cell interactions in many different cell types and

Leukemia Notch and Wnt signaling in T-cell development and T-ALL F Weerkamp et al 1200 Using a Tcf reporter construct, in which expression of a reporter gene is induced when Wnt signaling occurs in a cell, it was demonstrated that developing thymocytes can respond to Wnt signals in in vitro cultures.79 Recently, our group has shown that mainly DN1-3 thymocytes undergo Wnt signaling.82 Retroviral transduction of fetal thymocytes with Wnt1 and Wnt4 increased their proliferation in culture.79 Furthermore, blocking Wnt binding using soluble Fz receptors completely inhibited T-cell development at the DN and ISP stages.79 The requirement of Wnt proteins for thymocytes was confirmed in vivo, by showing that mice deficient for both Wnt1 and Wnt4 have twofold reduced thymic cellularity.83 This relatively mild reduction, combined with the fact that thymocyte subset distribution was normal in these mice, points out that different Wnt proteins are functionally redundant. The significance of Fz receptors in thymocytes was also indicated by the accelerated thymic atrophy observed in mice deficient for Fz9.84 But again the mild defect emphasizes the involvement of Figure 3 The canonical Wnt-signaling pathway Left: in the absence of Wnt binding, b-catenin is sequestered in the cytoplasm by the multiple Fz receptors in the thymus. tumor suppressor proteins Axin and adenomous polyposis coli (APC). Of the downstream Wnt-signaling components, Tcf-1 has b-Catenin present in this ‘destruction complex’, is phosphorylated by been most extensively studied. Two different Tcf-1 mutant mice serine/threonine kinases casein kinase (CK) and glycogen-synthase have been described, targeting either exon 5 (Tcf-1(V)) or exon 7 kinase 3b (GSK). Phosphorylated b-catenin is recognized by (Tcf-1(VII)).85 In Tcf-1(V) mutant mice, a low level of a truncated b-transducin-repeat-containing protein (b-TrCP), targeted for ubiquiti- yet functional Tcf-1 is still expressed, whereas the Tcf-1(VII) nation and degraded by the proteasome. In the absence of b-catenin, members of the Tcf family are bound in the nucleus by transcriptional mutation abolishes the DNA-binding activity of Tcf-1 comple- co-repressors (coR) of the groucho family. Right: Wnts bind to a tely and is therefore regarded as a true Tcf-1 null mutant. Young receptor complex consisting of a Frizzled (Fz) receptor and LRP5 or Tcf-1-deficient mice have an incomplete block at the DN1, LRP6. Wnt signals are transduced to the destruction complex via a yet DN2 and ISP stages, whereas older mice display a complete unknown pathway involving Dishevelled (Dsh), which may directly block at the DN1 stage of thymocyte development.85,86 interact with Fz. In addition, LRP may directly interact with Axin. Although Lef-1 knockouts have normal T-cell development,87 Signaling results in inhibition of GSK3b, which no longer can Lef-1/Tcf-1(V) double-deficient mice display a complete block phosphorylate b-catenin, allowing accumulation of b-catenin and its 88 translocation to the nucleus. b-catenin is targeted to the nucleus by in T-cell differentiation at the ISP stage, showing the Pygopus (Pygo) and Legless (LGS, Bcl-9). Binding of b-catenin to Tcf redundancy of these factors during thymocyte development. recruits histone acetylace CBP/p300 and Brg-1, a component of the The critical role of Tcf-1 in T-cell development may also lie in SWI/SNF chromatin remodeling complex. Several factors can mod- Wnt independent functions, for instance as transcriptional ulate Wnt signaling. For instance, interaction of Wnt with the receptor repressor. However, b-catenin binding to Tcf-1 is essential for complex can be prevented by Dickkopf (Dkk), which binds to the LRP co-receptor. Binding of b-catenin to Tcf/Lef can be inhibited by the thymocyte differentiation, as only Tcf isoforms which contain nuclear protein Chibby. the N-terminal b-catenin binding domain could rescue the thymic defect in Tcf-1(VII) mice.79,89 The importance of b-catenin for T-cell development is various species.70 The central player in the Wnt-signaling controversial. Presence of b-catenin mRNA was detected in all cascade (depicted in Figure 3) is the cytoplasmic protein thymoycte subsets.80 Mice with b-catenin deleted in all T cells b-catenin. In the absence of Wnt signaling, b-catenin is present from DN3 onwards showed impaired T-cell development at the in a cytoplasmic ‘destruction complex’ and is continuously b-selection checkpoint,90 but mice transplanted with HSCs in phosphorylated and degraded. When a Wnt protein binds to the which b-catenin had been inducibly deleted, did not show in receptor complex consisting of a member of the Frizzled (Fz) any thymic defect.91 As possible explanation, redundancy family of seven transmembrane proteins71 and an LDL-receptor- between b-catenin and its homologue plakoglobin (g-catenin) related protein (LRP5 or LRP6),72 b-catenin is no longer has been suggested. Irradiated mice reconstituted with phosphorylated, resulting in its accumulation and activation.73 plakoglobin-deficient fetal liver cells did not display marked Deposphorylated b-catenin translocates to the nucleus, where it defects in T-cell development, only a slightly reduced survival of activates Tcf/Lef transcription factors,74,75 in thymocytes pre- DN3 and DN4 thymocytes.92 Nevertheless, mice overexpres- dominantly Tcf-1.76,77 Next to the canonical Wnt-signaling sing Axin, one of the components of the b-catenin destruction pathway, b-catenin-independent mechanisms exist (reviewed by complex, exhibited reduced cell numbers and increased Veeman et al.78), but their implications for T-cell development apoptosis in their thymus.93 have not been studied in detail. What is the function of Wnt signaling during T-cell develop- In the thymus, Wnt proteins are mainly produced by the ment? The above-described studies of Tcf-1-deficient mice thymic epithelium, but to some extend also by the thymo- suggest that Wnt-signaling affects those stages of thymocyte cytes.79–82 Conversely, Fz receptors are expressed primarily by differentiation where proliferation occurs. By using several the thymocytes, but also by stromal cells.79,80,82 Redundancy methods to induce Wnt signaling in human CD34 þ thymocytes and promiscuous binding of different Wnts and Fz, together in combination with DNA microarrays, our group has identified with the difficulty to isolate biologically active recombinant Wnt target genes involved in proliferation and cell adhesion, but Wnt proteins, complicate the direct analysis of the effects of Wnt no T-cell-specific genes.94 Wnt target genes were shown to proteins on developing T cells. Nevertheless, numerous studies include c-fos and c-jun, which were indeed absent in ISP have now revealed functional roles for several Wnt-signaling thymocytes from Tcf-1-deficient mice.94 In addition, transcrip- components in T-cell development. tion of T-cell-specific genes and rearrangement of the TCRB

Leukemia Notch and Wnt signaling in T-cell development and T-ALL F Weerkamp et al 1201 genes was unaffected in Tcf/Lef-deficient mice,88 indicating that least T-cell-specific signals, for instance proliferative signals Wnt signaling does not directly induce a T-cell program. A role provided by preTCR signaling or other T-cell-specific prolif- for Wnt proteins in inducing proliferation would also fit with the erative signals (e.g. Wnt signaling to Tcf-1) are required to assumed function of Wnt signaling in stem cell self-renewal.94,95 induce T-ALL. A recent report indicates that suppression of p53 Nevertheless, a role for Wnt signaling in differentiation cannot by Notch may be the mechanism underlying deregulated T-cell be excluded, although it is difficult to distinguish between proliferation,104 whereas data in Notch3 transgenic mice proliferation and differentiation, as these processes are closely suggest that activation of the nuclear factor-kappaB pathway connected at this stage of T-cell development: massive by Notch may contribute to leukemogenesis.105 proliferation of DN1 and 2 thymocyte subsets is essential before further development can proceed. Similar to Notch signaling, additional roles for Wnt signaling Wnt in T-cell leukemia have been reported in more mature stages of thymocyte development. In DP and SP subsets, Wnts probably block Unlike Notch signaling, deregulated Wnt signaling has been apoptosis rather than inducing proliferation. In thymocytes in implicated in many forms of cancer.106 Two types of mutations which b-catenin was specifically activated from DN3 onwards, are found: inactivating (loss-of-function) mutations in the tumor apoptosis of thymocytes with non-productive TCRB rearrange- suppressor genes Axin and APC, or activating (gain-of-function) ments was reduced.96 Using the same approach but with mutations in b-catenin. Surprisingly, considering the unmistak- lower levels of b-catenin, it was shown that constitutive able role of Wnt signaling during T-cell development, no b-catenin signaling enhanced the generation of mature thymo- involvement in T-ALL has been described thus far. Such a role is cytes, interestingly preferentially of CD8 þ cells.97 In accor- highly likely and may be caused by activating mutations in dance, deletion of b-catenin at the same stages impaired positive regulators of the Wnt-signaling pathways. Preliminary T-cell development at the b-selection checkpoint.90 data from our laboratory indicate a fairly high frequency of Tcf-1-deficient DP thymocytes were demonstrated to die more T-ALL samples with accumulation of nuclear b-catenin, a rapidly than wild-type cells, probably because of lower hallmark of active Wnt signaling. We are currently exploring 89 expression of antiapoptotic protein Bcl-xL. Recently, it was these findings in more detail. shown that survival of pre-TCR þ thymocytes requires the Little by little, data are now accumulating to support a b-catenin binding domain of Tcf-1.92 causative role for Wnt signaling in other hematopoietic malignancies. Perhaps the most striking evidence is provided by a seminal paper by Weissman et al. reporting involvement of Notch in T-cell leukemia Wnt signaling in the proliferation of chronic myelogenous leukemia (CML) stem cells.107 Using a lentiviral TCF-GFP Animal models have demonstrated a role for Notch in T-cell reporter, Weissman and colleagues provided evidence that leukemias.38,98,99 But although the t(7;9)(q34;q34.3) transloca- Wnt signaling is activated in blast crises of CML. Other reports tion, in which part of NOTCH1 is fused to the TCRB locus, was indicate involvement of Wnt signaling in AML, CLL and multiple observed in rare cases of T-ALL already in 1991,4 scientists only myeloma.108–111 Studies on ALL are conspicuously absent, recently became aware of the tremendous importance of except for one report showing that the E2A-PBX1 fusion protein, Notch1 for T-cell leukemogenesis in humans. Translocation involved in certain types of precursor B-ALL, activates the (7;9) results in aberrant Notch1 expression (originally termed expression of Wnt16.112 But studies with patient material and TAN1 onocogene) and is found in less than 1% of T-ALL. comparisons with normal precursor B cells (which undergo Wnt Weng et al.100 discovered that over 50% of human T-ALL has signaling) are lacking. activating mutations in Notch1. This important finding was Interestingly, in apparent contrast to the fact that enhanced the result of the observation that several T-ALL cell lines not Wnt signaling causes cancer, Tcf-1 in absence of b-catenin also harboring translocation (7;9), were sensitive to g-secretase functions as a tumor suppressor. Tcf-1-deficient mice develop 113 inhibitors, resulting in a G0 cell cycle arrest. Sequencing of adenomas in the gut and mammary glands and ageing the NOTCH1 gene in these cell lines resulted in identification animals develop a T-cell hyperplasia with all characteristics of two types of mutations. These mutations occur either in the of human T-ALL (Baert et al., manuscript in preparation). heterodimerization domain, causing an instable connection Wnt5a hemizygous mice develop myeloid leukemias and B-cell between the intracellular and extracellular domain, or in the lymphomas,114 confirming a role for Wnt signaling in suppres- negative regulatory PEST domain. Both mutations lead to sion of proliferation, presumably through the non-canonical increased levels of Notch-dependent transcription in reporter Wnt pathway. assays, presumably by increasing nuclear Notch1 levels.100 The same types of mutations were found in a panel of primary T-ALL samples. Interestingly, these mutations were not associated with Notch and Wnt signaling: coalition or opposition? a particular subtype of T-ALL and occurred in all major subtypes of T-ALL, both in pediatric and adult patients.100–102 Obviously, It is generally thought that Wnt and Notch fulfill opposing these findings have therapeutic implications, as g-secretase functions in the thymus: Wnt in proliferation, Notch in differen- inhibitors could be used to treat T-ALL. However, given that tiation, and that these processes are mutually exclusive. Notch signaling also is implicated in many other developmental However, it may well be possible that Wnt and Notch signals processes, including HSC self-renewal, such approaches need to combine forces to induce proliferation of the earliest thymo- be implemented with great caution. cytes. Gain-of-function mutation in positive effectors in the It is incompletely understood why ectopic expression of Notch and Wnt pathway cause cancer, indicating that both may IC-Notch leads to leukemia. Data in the mouse indicate that lead to increased proliferation. IC-Notch must cooperate with pre-TCR signaling to cause Both Wnt115 and Notch116,117 signaling have been implicated leukemia, as no T-cell development and T-ALL occurred when in HSC self-renewal. A recent report has suggested molecular IC-Notch was over expressed in RAGÀ/À or SLP76À/À BM.103 At cooperation between the Notch and Wnt pathways: Notch

Leukemia Notch and Wnt signaling in T-cell development and T-ALL F Weerkamp et al 1202 3 Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 1982; 31: 99–109. 4 Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991; 66: 649–661. 5 Anderson G, Jenkinson EJ. Lymphostromal interactions in thymic development and function. Nat Rev Immunol 2001; 1: 31–40. 6 Bhandoola A, Sambandam A, Allman D, Meraz A, Schwarz B. Early T lineage progenitors: new insights, but old questions remain. J Immunol 2003; 171: 5653–5658. 7 Weerkamp F, Baert MR, Brugman MH, Dik WA, de Haas EF, Visser TP et al. The human thymus contains multipotent progenitors with T/B-lymphoid, myeloid and erythroid lineage potential. Blood 2006; 107: 3131–3137. 8 Scollay R, Bartlett P, Shortman K. T cell development in the adult Figure 4 Hypothetical order of events during the earliest stages of murine thymus: changes in the expression of the surface antigens T-cell development. The thymus-seeding cells (TSCs) encounter Ly2, L3T4 and B2A2 during development from early precursor Delta ligands on the thymic stroma and start their development into cells to emigrants. Immunol Rev 1984; 82: 79–103. the T-cell lineage. These early thymocytes rapidly upregulate Tcf1, 9 Weerkamp F, de Haas EF, Naber BA, Comans-Bitter WM, Bogers which is subsequently activated by Wnt signals. Wnt target genes (e.g. AJ, van Dongen JJ et al. Age-related changes in the cellular c-fos and c-jun) and Notch target genes (e.g. Hes1) together induce composition of the thymus in children. J Allergy Clin Immunol massive proliferation of early thymocytes. 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