(2013) 27, 541–552 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

REVIEW B-lineage factors and cooperating required for leukemia development

E Tijchon, J Havinga, FN van Leeuwen and B Scheijen

Differentiation of hematopoietic stem cells into B requires the concerted action of specific transcription factors, such as RUNX1, IKZF1, E2A, EBF1 and PAX5. As key determinants of normal B-cell development, B-lineage transcription factors are frequently deregulated in hematological malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and affected by either chromosomal translocations, gene deletions or point . However, genetic aberrations in this developmental pathway are generally insufficient to induce BCP-ALL, and often complemented by genetic defects in receptors and tyrosine kinases (IL-7Ra, CRLF2, JAK2 and c-ABL1), transcriptional cofactors (TBL1XR1, CBP and BTG1), as well as the regulatory pathways that mediate cell-cycle control (pRB and INK4A/B). Here we provide a detailed overview of the genetic pathways that interact with these B-lineage specification factors, and describe how mutations affecting these master regulators together with cooperating lesions drive leukemia development.

Leukemia (2013) 27, 541–552; doi:10.1038/leu.2012.293 Keywords: BCP-ALL; focal deletions; chromosomal translocations; mutations; transcription factors; cooperative

INTRODUCTION recurrent point mutations, focal gene deletions and rearrange- 2–4 Acute lymphoblastic leukemia (ALL) is the most common form of ments. In general, BCP-ALL harbors gene lesions that affect in children characterized by a block in lymphoid transcription factors and signaling molecules implicated in differentiation, leading to accumulation of immature progenitor lymphoid differentiation, such as IKZF1, RUNX1, TCF3, EBF1, PAX5, cells in the , peripheral and occasionally the VPREB1 and RAG1/RAG2, as well as that control cell central . Although ALL cure rates are approaching survival and proliferation, including ABL1, NRAS/KRAS, JAK2, IL7R 90%, it remains the leading cause of cancer-related mortality in and CRLF2 (Table 1). Mutations in these two common pathways children and young adults. The most prevalent form of childhood are often combined with genetic defects in transcriptional ALL is B-cell precursor ALL (BCP-ALL), which represents about 85% coregulators, such as TBL1XR1, CREBBP, BTG1 and well-known of the cases, whereas the remaining 15% of the cases involve tumor-suppressor involved in regulation of the , T-lineage ALL (T-ALL). In BCP-ALL, two variants of , such as RB1, CDKN2A, CDKN2B and TP53. Surprisingly, however, namely hyperdiploidy of more than 50 and many of the BCP-ALL genetic subtypes display a rather unique hypodiploidy, as well as a collection of recurrent chromosomal pattern of specific cooperating gene lesions. rearrangements, give rise to distinct genetic subtypes.1 These In this review, we will focus on the key hematopoietic rearrangements include commonly affected genes, such as MLL, transcription factors targeted in BCP-ALL, describing their role in CRLF2 or c-, fused to a diverse range of different partner genes normal and malignant hematopoiesis, and summarizing the or regulatory elements, and uniquely defined chromosomal cooperating gene lesions and the critical transcriptional targets/ translocations, such as BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1. pathways that drive leukemia formation. Specifically, we describe Some of these genetic subtypes, such as ETV6-RUNX1 and in detail the posttranslational modifications and cofactors that are hyperdiploidy, which together include about 50% of the cases, associated with these transcription factors, regulating their activity correlate with a favorable outcome, whereas others, like the MLL and stability. A more precise understanding of the rearrangements, BCR-ABL1 and hypodiploidy, covering about 13% regulatory pathways that control these of the cases, are associated with a poor treatment response.1 functions and their cooperating signaling molecules may hold Many of these chromosomal rearrangements constitute ‘driver’ the key to development of novel targeted therapies aimed at mutations and can be detected in both diagnosis and relapse specific BCP-ALL subtypes. samples. However, it is evident from experimental mouse models and through extensive genomic profiling of patient samples that these genetic driver lesions are usually insufficient to induce RUNX1 leukemia and require cooperating events. Both single-nucleotide One of the most frequently mutated genes in leukemia is RUNX1 polymorphism arrays, as well as deep-sequencing of genomes, (encoding the (AML) 1 protein), which and transcriptomes have provided important new insights into belongs to the Runt-related family of transcription factors, the different cellular pathways that are commonly affected by together with RUNX2 (AML3) and RUNX3 (AML2). The RUNX

Laboratory of Pediatric Oncology, Radboud University Medical Centre, Radboud University Centre of Oncology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands. Correspondence: Dr B Scheijen, Laboratory of Pediatric Oncology, Radboud University Medical Centre, Radboud University Centre of Oncology, Nijmegen Centre for Molecular Life Sciences, Trigon Building Room 276, Route 224, P.O. Box 9101, 6500 Nijmegen, The Netherlands. E-mail: [email protected] Received 24 July 2012; revised 26 September 2012; accepted 27 September 2012; accepted article preview online 10 October 2012; advance online publication, 23 November 2012 B-lineage transcription factors in leukemia E Tijchon et al 542 Table 1. Functional classification of the genes affected in BCP-ALL

Pathwaysa Genes

Lymphoid differentiation RUNX1, ERG1, IKZF1, IKZF2, IKZF3, TCF3, TCF4, TCF12, EBF1, PAX5, LEF1, TCF7L2, ETV6, SPI1, MEF2C, IRF8, C-MYC, NR3C1, NR3C2, RAG1, RAG2, VPREB1, CD200/BTLA Proliferation and cell survival ABL1, JAK1, JAK2, JAK3, KRAS, NRAS, IL7R, CRLF2, FLT3, PTPN11, NF1 Cell-cycle regulation CDKN2A, CDKN2B, RB1, TP53 Transcriptional cofactors TBL1XR1, BTG1, CREBBP, NCOR1, BCOR, CTCF, ARID5B Others FHIT, C20orf94, ASMTL, MUC4, ADARB2, ADD3, ARPP21, DMD Abbreviation: BCP-ALL, B-cell precursor acute lymphoblastic leukemia. aIn BCP-ALL, most of the genes that are affected by chromosomal rearrangements, focal gene deletions and point mutations in diagnosis and relapse samples can be classified into four different genetic pathways.1–4

proteins together with the common core-binding factor-beta (CBFb) make up the heterodimeric CBF transcription factors, which have important roles in hematopoiesis and bone development. The RUNX proteins directly bind DNA via an N-terminal runt homology domain that also mediates heterodimerization with CBFb, which is required for efficient binding to the RUNX RUNX1 Primitive consensus site and results in activation or repression of target HSC genes. The RUNX1 gene generates three alternatively spliced variants, giving rise to RUNX1a, RUNX1b and RUNX1c, each of which possesses the DNA-binding domain (DBD), while RUNX1a lacks the C-terminal transcriptional regulatory domain. RUNX1 can ERG recruit a variety of transcriptional coactivators, such as the Definitive acetyltransferases MOZ and p300/CBP,5,6 the transcriptional HSC CLP Pre-Pro B Pro-B Pre-B regulator YAP1 and the protein subunits BRG1 and INI1, which are components of the adenosine triphosphate (ATP) depen- 7 dent SWI/SNF -remodeling complex. Transcriptional IKZF1 E2A PAX5 repression by RUNX1 is mediated through direct binding to EBF1 the histone deacetylases HDAC1 and HDAC3, the histone methyltransferase SUV39H1 and recruitment of the SIN3A and 8,9 Figure 1. Schematic overview of the normal function of the different TLE/Groucho complexes via the C-terminal region. B-lineage transcription factors affected in BCP-ALL. B-lineage In addition, RUNX1 can interact in a cooperative manner with transcription factors affected in BCP-ALL often act at different several tissue-specific transcription factors, such as GATA1, ETS1, stages of hematopoietic development, but their earliest critical MYB, C/EBPa, PU.1 and STAT5, thereby able to modulate the function in hematopoiesis as revealed by gene-knockout mouse expression of a broad range of transcriptional target genes. studies is indicated. RUNX1 is required for the emergence of RUNX1 can be modified through phosporylation by the MAP primitive HSCs during , whereas ERG is kinases ERK1/2,10 which results in enhanced by essential for definitive HSC maintenance. IKZF1 expression is necessary for the formation of common lymphoid progenitors RUNX1. In addition, cyclin-dependent kinases (CDKs) are able to (CLPs), and E2A and EBF1 are essential to generate pro-B cells. PAX5 phosphorylate RUNX1 on multiple sites, thereby regulating fulfils a critical role in maintaining B-cell identity, and its expression different aspects of AML1 function, including its degradation is required to generate functional pre-B cells. LEF1 mainly regulates during G2/M phase of the cell cycle by the anaphase-promoting early B-cell survival signaling and proliferation. complex,11 and the interaction with HDAC1 and HDAC3.12 The DNA-binding affinity of RUNX1 is enhanced by p300-mediated at two lysine residues (K24 and K43 in RUNX1b).6 In contrast, both DNA-binding and transactivating properties of progenitors in the bone marrow19 (Table 2). The identification of RUNX1 are abrogated through lysine by SUV39H1,13 RUNX1 target genes has provided some insight into its function whereas arginine methylation of residues R206 and R210 by during hematopoiesis. Runx1 insufficiency suppresses protein arginine methyltransferase 1 (PRMT1) disrupts SIN3A- and delays cell-cycle progression in the stem/ mediated transcriptional repression of RUNX1.14 RUNX1 function is fraction, which is accompanied by altered expression of the anti- also modulated by PRMT4/CARM1, which induces R223 apoptotic protein Bcl2 and the polycomb ring-finger oncoprotein methylation and binding of the zinc-finger protein DPF2, Bmi1.20 The PU.1 (Sfpi1) gene is an essential target of RUNX1 resulting in differential target-gene regulation.15 Furthermore, expression in all three major hematopoietic lineages. In myeloid PRMT5, in complex with its COPR5, regulates nuclear and T-lymphoid cell lineages, RUNX1 regulates the Il3, Csf2, Csf1r, export of RUNX1 through R142 methylation.16 Cd4 and Tcrd genes, whereas in B-lineage differentiation, RUNX1 is RUNX1 and CBFb are essential for all lineages of definitive essential for Ebf1 expression (Figure 2).21 Mouse models have hematopoiesis in mouse fetal liver, including the formation of demonstrated that both RUNX1 gain-of-function and loss-of- erythroid cells, , and lymphoid function predisposes to hematological malignancies. Enhanced or cells.17,18 In particular, mouse Runx1 expression is required for deregulated expression of Runx1 due to integration of murine the emergence of hematopoietic stem cells (HSCs) from the leukemia virus is a recurrent event upon proviral tagging of vascular in the midgestation embryo (Figure 1). In -prone mice, and RUNX1-transgene expression contrast, conditional gene-targeting experiments have revealed accelerates the onset of B- and T-cell of Em-Myc that the Runx1 gene is not essential for the maintenance of adult transgenic mice.22 On the other hand, conditional or chimeric long-term HSCs during definitive hematopoiesis. Rather, Runx1 Runx1-deficient mice develop thymic lymphomas,23,24 and are deficiency blocks megakaryopoiesis and develop- predisposed to leukemia development upon retroviral insertional ment, while at the same time expands multipotent and myeloid mutagenesis.25

Leukemia (2013) 541 – 552 & 2013 Macmillan Publishers Limited B-lineage transcription factors in leukemia E Tijchon et al 543 Table 2. of mice harboring altered function of B-lineage transcription factors

Transcription Phenotype of knockout mouse models Lymphoid malignancies in mouse models factor (gene)

RUNX1 Runx1 À / À : embryos die around E12.5; no primitive and definitive ETV6-RUNX1 knock-in: expansion of HSCs, block in (Runx1) hematopoiesis17 B-cell development, no B-cell malignancies33 Runx1 conditional knockout: impaired megakaryopoiesis and lymphocyte Runx1 conditional knock out: develop T-cell development; expansion of myeloid progenitors19 malignancies24 ERG (Erg) ErgMld2/Mld2: embryos die at E11.5–12.5; no definitive hematopoiesis36–38 Erg transgenic: develop T-ALL40 ErgMld2/ þ : defective stress-induced HSC function, impaired B-cell development and megakaryopoiesis36–38 IKZF1 (Ikzf1) IkarosL/L: absence of pDCs and a reduction in pre-pro-B cells52 Ikaros-null À / À : mild reduction in HSCs and absence of B cells, NK cells and Ikaros-null þ / À : develop thymic lymphomas with a fetal T cells53,54 low incidence Ikaros-DN À / À : strong reduction in HSCs and no B cells, T cells, NK cells and Ikaros-DN þ / À : develop thymic lymphomas DCs56 Ikaros-Plastic À / À : embryos die around E16.5; block in erythropoiesis and Ikaros-Plastic þ / À : develop thymic lymphomas lymphopoiesis57 E-box- Tcf3 À / À : impaired T lymphopoiesis and block at the pre-pro-B-cell Tcf3 À / À : develop T-cell lymphomas80 binding stage78,79 proteins E2A (E12/ E47 À / À : block at the pre-pro-B-cell stage79 TCF3-PBX1: develop T-cell lymphomas and E47) (Tcf3/ myeloid Tcfe2a) E12 À / À : impaired B-cell differentiation79 TCF3-HLF: develop T-cell lymphomas E2-2 (Tcf4) Tcf4 À / À : are born with extremely low frequency and die around birth. Mild Tcf4 À / À : no lymphoma/leukemia reduction in the number of pro-B cells and no pDCs81–83 HEB (Tcf12) Tcf12 À / À : die within 2 weeks after birth. Mild reduction in the number of Tcf12 À / À : no lymphoma/leukemia pro-B cells81 EBF1 (Ebf1) Ebf1 À / À : block at the pre-pro- stage99 Ebf1 þ / À and Ebf1 À / À : no spontaneous hematological malignancies Ebf1 þ / À : reduction in the number of pro-B and splenic B-cells99 PAX5 (Pax5) Pax5 À / À : early block in B-cell differentiation during fetal hematopoiesis. In Pax5 transgenic: develop T-cell lymphomas adult bone marrow, block at the pro-B-cell stage with no VH–DJH recombination116–117 Pax5 conditional knockout: conversion of mature B cells into functional T Pax5 conditional knock out: Develop progenitor cells through dedifferentiation to uncommitted progenitors123 B-cell lymphomas123 LEF1 (Lef1) Lef1 À / À : reduced proliferation and enhanced apoptosis of pro-B cells; no Lef1 þ / À and Lef1 À / À : no spontaneous defect in B-cell differentiation135 hematological malignancies Abbreviations: HSC, hematopoietic ; pDC, plasmacytoid dendritic cell; DN, dominant-negative; T-ALL, T-lineage ALL.

In , deregulation of RUNX1 expression through chro- gene deletions in NR3C1, NR3C2, BTG1 and TBL1XR1 (Figure 3a)3, mosomal translocations, mono- and bi-allelic deletions, point which components and regulators of nuclear receptors mutations and gene amplification is observed in multiple and together are present in B45% of the ETV6-RUNX1 cases. In hematopoietic malignancies, including AML, ALL, chronic myeloid addition, 70% of the ETV6-RUNX1-positive leukemias harbor focal leukemia and . In addition to somatic deletions in the pre-B-cell gene VPREB1. Another mutations, germline point mutations in RUNX1 are responsible for characteristic feature of ETV6-RUNX1-positive leukemias is the an autosomal-dominant disorder with a propensity to high frequency of ETV6 deletions affecting the other develop leukemia (Familial platelet disorder (FPD)-AML).26 The nontranslocated ETV6 allele in about three quarters of BCP-ALL most prevalent translocations involving the RUNX1 gene located cases, arguing that loss of TEL function cooperates with ETV6- on 21q22.12 are RUNX1-MTG8 (encoding the AML1- RUNX1 in leukemogenesis. ETO protein), which occurs in about 15% of AML cases, and ETV6- The ETV6-RUNX1 has retained the N-terminal RUNX1 (encoding the TEL-AML1 protein) that represent the most non-DNA-binding region of TEL containing the oligomerisation common genetic subtype in pediatric BCP-ALL, present in 25% of pointed domain, as well as the central repression domain, the cases. Although the ETV6-RUNX1 translocation is associated together with all functional domains of RUNX1 (Figure 4a). The with a favorable outcome, intrachromosomal amplification of prevailing notion is that ETV6-RUNX1 binds RUNX1 target leading to RUNX1 gene amplification predicts an sequences and acts as a constitutive inhibitor of transcription increased risk of relapse and is present in about 2% of childhood through recruitment of SIN3A- and NCoR1-containing corepressor BCP-ALL cases.27 The ETV6-RUNX1 translocation arises most often complexes by the ETV6 part of this fusion protein. ETV6-RUNX1 in utero, generating a preleukemic clone that can persist for years has been shown to promote B-cell differentiation and enhance the before overt leukemia develops, and which requires additional self-renewal of multipotent and B-cell progenitors in culture,28,29 cooperating events. These include deletions in the CDKN2A which requires DNA- and CBFb-binding of the runt homology and copy number alterations (CNAs) in genes regulating B-cell domain of RUNX1.29 Furthermore, ETV6-RUNX1 interferes with differentiation, such as PAX5 (28%) and EBF1 (10%), but not IKZF1.3 apoptosis through regulation of survivin via the microRNAs miRNA- Furthermore, there is a very strong enrichment for cooperating 494 and miRNA-320a. Other specific pathways affected by

& 2013 Macmillan Publishers Limited Leukemia (2013) 541 – 552 B-lineage transcription factors in leukemia E Tijchon et al 544

Figure 2. Gene regulatory interactions of B-lineage transcription factors at the early stages of B-cell development. B-lineage specification starts at the lymphoid-primed multipotent progenitor stage, and continues in the CLP and pro-B cell stage. B-cell differentiation is regulated by several transcription factors, including RUNX1, IKZF1, EBF1, E2A and PAX5, that cross-regulate each other at the transcriptional level. Furthermore, the B-lineage transcription factors induce expression of the critical cytokine receptors FLT3 and IL7R, as well as target genes that are important for VDJ recombination, pre-B-cell receptor signaling and B-lineage commitment. Transcriptional targets that fulfill no active role in signaling/transcription regulation at the specified stage are indicated in italic and gray, while cytokine receptors are boxed to distinguish them from B-lineage transcription factors.

ETV6-RUNX1 expression include the phosphoinositide 3-kinase/ maintenance, ERG is essential to drive expression of Runx1 and AKT/mammalian target of rapamycin (mTOR) and TGF-b Gata2 and to prevent stem cell exhaustion.37 ErgMld2/ þ mice show pathways.30,31 An ETV6-RUNX1 knock-in allele or transgenic adequate steady-state HSC maintenance, but display defective expression of ETV6-RUNX1 is insufficient to induce leukemia in HSC renewal during stress-induced hematopoiesis, as well as mice,32,33 but may predispose ETV6-RUNX1 mice to carcinogen- defective megakaryopoiesis and B-cell development in induced T-lineage malignancies.33 Similarly, transplantation of competitive repopulation assays.38 Retroviral expression of ERG fetal liver-derived progenitor cells expressing ETV6-RUNX1 in progenitor cells appears to promote megakaryopoiesis, increase into lethally irradiated recipient mice yields no leukemia, but the frequency of myeloid progenitors and induce expansion of T confers a selective advantage on hematopoietic repopulation.28 cells, pro-B cells and erythroid cells. Moreover, enforced ERG Hence, future experiments will need to identify the key expression can induce megakaryoblastic leukemia and T-ALL in cooperating events that drive ETV6-RUNX1-induced progenitor mice, but not B-lineage leukemia.39,40 B-cell leukemia development. One promising candidate is the Deregulation of ERG function is observed both in solid tumors, tumor-suppressor gene CDKN2A, which has been shown to such as , Ewing’s sarcoma and primitive neuroec- promote ETV6-RUNX1-induced leukemogenesis in a bone todermal tumors, and in hematological malignancies.41 In marrow transplantation assay.34 leukemia, ERG expression can be downregulated through miR- 196a and miR-196b. The chromosomal translocation FUS-ERG is detected in AML and BCP-ALL,41,42 while overexpression of ERG is ERG associated with poor prognosis in T-ALL, cytogenetically normal The ERG gene encodes a member of the E-twenty-six (ETS) family adult AML and MLL-rearranged childhood AML. ERG has also been of transcription factors, which are characterized by an ETS domain implicated in the development of transient myeloproliferative encoding a winged helix-turn-helix structure for DBD and a disorder and acute megakaryocytic leukemia in pointed protein-interaction domain. The ERG gene encodes six patients, as ERG is located on chromosome 21q22.2.43 This is different protein isoforms, due to distinct transcriptional start and supported by a mouse model for Down syndrome-induced termination sites, as well as events. The ERG myeloproliferation, where the Erg þ /Mld2 allele corrects the subfamily also includes ETS family members FLI1 and FEV, both of hematological features of this malignant disorder.44 which have been implicated in different forms of cancer. Many ETS Childhood BCP-ALL is characterized by the presence of ERG proteins are phosphorylated in response to growth-factor point mutations and mono-allelic gene deletions, which are stimulation and cellular stress, but the exact residues modified predicted to produce a truncated ERG protein.3 Recent studies in ERG as well as the responsible kinases remain to be identified have defined ERG gene deletions as a separate genetic subtype and investigated in more detail. By employing high-throughput present in childhood ALL (±7%) associated with a unique gene- sequencing of ERG-binding sites in hematopoietic progenitor cells expression cluster, which correlates with a good treatment after chromatin immunopreciptation, it has become evident that outcome,45,46 although gain of ERG mutations have been ERG binds not only to canonical ETS sites but also shows direct described in relapsed BCP-ALL cases. This and indirect cooperative interactions with other transcriptional cluster is characterized by a high frequency of NRAS gene regulators, including GATA2, SCL, LYL1, RUNX1, LMO1 and FLI1.35 mutations. ERG is a transcriptional regulator of the noncanonical The ErgMld2/Mld2 mouse mutant harboring a loss-of-function Erg WNT11 gene47 and the serine/threonine protein kinase PIM1,48 but allele, which fails to induce target-gene expression, has revealed it remains to be established which gene targets of ERG are critical that ERG fulfills a critical role in sustaining definitive hemato- in BCP-ALL. poiesis36,37 (Figure 1). During early embryonic hematopoietic development, Erg is not required for the initial hematopoietic lineage specification from the mesoderm, which coincides with IKZF1 the essential function of Runx1 in HSC emergence. However, at the Since its discovery as a key regulator of lymphopoiesis, the second wave of hematopoietic development, which involves HSC transcription factor IKZF1 (Ikaros) and its family members, encoded

Leukemia (2013) 541 – 552 & 2013 Macmillan Publishers Limited B-lineage transcription factors in leukemia E Tijchon et al 545

CRLF2 IL7Rα

JAK

BCR ABL

Transcriptional co-factors

BTG1 TBL1XR1 Transcription IKZF1

Transcriptional repression

GR ETV6 RUNX1 Proliferation Survival

Figure 3. Differential requirement for distinct cooperating events in specific genetic subtypes of BCP-ALL. (a) ETV6-RUNX1-positive leukemias display a selective enrichment for gene lesions in transcriptional cofactors BTG1 (19% versus 5% in the remaining BCP-ALL subtypes) and TBL1XLR1 (13% versus 1% in the remaining BCP-ALL subtypes) together with genetic aberrations in nuclear hormone receptors, mainly the (GR), encoded by the gene NR3C1 (13% versus 2% in the remaining BCP-ALL subtypes), as well as the mineralocorticoid receptor, encoded by the gene NR3C2 (11% versus 1% in the remaining BCP-ALL subtypes).3 (b) Focal gene deletions and point mutations affecting the tumor-suppressor IKZF1 frequently co-occur with gain-of-function mutations in tyrosine kinases, such as BCR- ABL1, JAK2 and cytokine receptors CRLF2 and IL-7Ra. Solid lines indicate direct physical interactions, while dotted lines indicate genetic interactions, that is, cooperation in leukemogenesis.63,65,66.

by the genes IKZF1-IKZF5, have been the subject of intense study in phenotype with absence of peripheral plasmacytoid dendritic cells the field of immunology.49 These transcription factors are and a reduction in pre-pro-B cells, but relatively normal numbers of characterized by an N-terminal conserved zinc-finger domain that mature, polyclonal B cells.52 On the other hand, Ikaros-null À / À mediates DNA binding, and a C-terminal zinc-finger domain mice show a 30-fold decrease in HSC activity and are completely required for homo- and/or heterodimerization with IKZF family deficient in B cells, natural killer cells and fetal T-cells.53,54 members. Alternative splicing of the IKZF1 gene can generate 13 Postnatally, a reduced number of T-cells differentiate in the different isoforms that vary in the number of N-terminal zinc fingers thymus, but these T cells display defects in CD4- versus CD8- and often display compromised DNA binding, giving rise to lineage differentiation and pre-T-cell receptor and T-cell-receptor dominant-negative (DN) isoforms. Like the RUNX1 protein, IKZF1 selection.55 A more severe phenotype is observed in mice is known to act both as a and as an activator of expressing a DN isoform, which lacks a functional DBD but still transcription. IKZF1 represses target genes through recruitment of contains an intact dimerization motif allowing it to homo- or HDAC-dependent (SIN3A and Mi-2b/NuRD complexes) and HDAC- heterodimerize (Ikaros-DN). Ikaros-DN mice display a more than independent (CtIP and CtBP) .50 Transcriptional 100-fold reduction in HSC activity and a complete lack of B-cells, activation by IKZF1 involves interaction with BRG1-containing T-cells, natural killer cells and all types of DCs.56 The most severe SWI/SNF chromatin-remodeling complexes, and through transcription elongation via recruitment of the P-TEFb component phenotype has been described for mice with a single amino-acid Cdk9.50,51 IKZF1 function can be modified through several change (His191Arg) in the third zinc finger of the DBD, called Ikaros- posttranslational modifications. Phosphorylation of IKZF1 protein Plastic, which interferes with the function of all IKZF family by casein kinase 2 at multiple sites affects DNA binding, localization members. Mice homozygous for this die between embryonic day E15.5 and E17.5 due to an additional block in to pericentromeric heterochromatin and controls degradation via 57 the ubiquitin pathway.50 Furthermore, SUMOylation of IKZF1 at two erythroid differentiation resulting in severe . In contrast, þ / À lysine residues (K58 and K241) disturbs its function as Ikaros-null mice show rather normal lymphoid differentiation, / / transcriptional repressor.50 and especially the Ikaros-DN þ À and Ikaros-Plastic þ À animals are IKZF1 has a critical role at the early stages of hematopoiesis and highly susceptible to develop T-cell malignancies. Consistent with lymphoid commitment as shown in different mouse models. Mice these observations, the Ikzf1 gene is frequently subjected to genetic homozygous for a hypomorphic Ikzf1 allele (IkarosL/L), harboring a alterations in various types of mouse T-cell lymphomas, including beta-galactosidase reporter within the Ikzf1 gene, display a mild mutations, deletions and inactivating retroviral insertions.58

& 2013 Macmillan Publishers Limited Leukemia (2013) 541 – 552 B-lineage transcription factors in leukemia E Tijchon et al 546 ETV6 RUNX1

RHD TRD

8 23 4 5 6 7 9 ATG 1359bp

L117fs IKZF1 I110fs G158S H224fs G337fs S402fs E504fs R111

1 2 3 4 5 6

DNA binding domain Dimerization domain

Exons 23 4 5 6 7 8 ATG 1557bp ∆4-8 ∆4-7 (DN Ik6) ∆4-6 (DN Ik8) ∆2-7 ∆2-8

PBX1 E2A HLF

AD1 AD2 HLH

Exons 2 3 4 5 6 7 8 910 11 12 1314 15 16 17 18 19 ATG 1962bp

EBF1 DBD IPT/TIG HLH

Exons 1 234 5678 9 10 11 12 13 14 15 16 ATG 1773bp Δ8 Δ7-9 Δ1-10 Δ1-14

JAK2 PAX5 BRD1 HIPK1 FOXP1 NCoR1 ELN DACH1 PML DACH2 AUTS2 ZNF521 ETV6 POM121 GOLGA6 C20orf112 R59G T75R D53V V319fs P80R V151T E201fs I301T P320fs G24R V336fs

PD O H TRD

Exons 1 2 3 4 5 6 7 8 9 10 ATG 1173bp Δ2-3 Δ2-5 Δ2-6 Δ2-7 Δ2-8 Δ6-8 Δ6-7 Δ8 Figure 4. Overview of the type of genetic lesions that affect B-lineage transcription factors. (a) The RUNX1 gene comprises of nine exons and an open-reading frame of 1359 bp. The chromosomal translocation ETV6-RUNX1 confers RUNX1 into a constitutive transcriptional repressor, which is dependent on the Runt homology domain (RHD) for DNA binding. The transcriptional regulatory domain (TRD) of RUNX1 still contributes to the recruitment of corepressor proteins. (b) The IKZF1 gene, which consists of eight exons, is targeted by point mutations generating frameshift (fs) or missense mutations, as well as mono-allelic deletions affecting a larger portion of the gene (D4–8) without producing a functional protein (’—’), and intragenic deletions (2) generating DN isoforms (D4–6 and D4–7). These DN isoforms lack the DBD that comprises of four zinc fingers, but retain the dimerization domain, which consists of two zinc fingers. (c) The TCF3 gene encoding the E2A protein consists of 19 exons. The chromosomal translocations TCF3-PBX1 and TCF3-HLF retain the transcriptional activation domains (ADs) of E2A, but harbor the DBD of PBX1 and HLF, respectively. The HLH domain of E2A is not present in the oncogenic fusion proteins. (d) The EBF1 gene, which consists of 16 exons, is exclusively affected by focal gene deletions that cover a large portion of the coding region, including the ATG (’—’), or generate truncated proteins (’-) encoding a part of the DBD, but lack the IPT/TIG and helix-loop-helix-loop- helix motif. (e) The PAX5 gene, which consists of 10 exons, is affected by different gene lesions, including missense and frameshift mutations, intragenic deletions often giving rise to truncated proteins that lack the paired domain (PD) required for DNA binding and translocations. These translocations with multiple-partner genes produce oncogenic PAX5 fusion proteins that still harbor the PD domain, but may lack the octapeptide (O) involved in Groucho binding, the homeodomain-like region (H) and TRD, depending on the breakpoint of the translocation.

Leukemia (2013) 541 – 552 & 2013 Macmillan Publishers Limited B-lineage transcription factors in leukemia E Tijchon et al 547 The essential role of IKZF1 in hematopoiesis is demonstrated by E-BOX-BINDING PROTEINS the fact that IKZF1 primes lymphoid development in the HSC by Transcriptional regulators of the E-box-binding activating transcription of lymphoid-associated transcription have important roles in neuronal, muscle cell and lymphoid factors (Ebf1) and signaling molecules (Flt3 and IL7r) (Figure 2), differentiation. E proteins E12 and E47, generated through while simultaneously suppressing myeloid-associated transcrip- alternative splicing of the E2A (TCF3) gene, constitute the class I tion factors (Sfpi1 and Gfi1). Second, IKZF1 shuts down gene helix-loop-helix (HLH) transcription factors, together with E2-2 transcription programs in the lymphoid-primed multipotent (TCF4) and HEB (TCF12). These HLH transcription factors are progenitors that correlate with stemness (for example, Gata2 characterized by a basic region that mediates DNA binding to the and Thy1) and multipotency (for example, Gata1). IKZF1 controls E-box-recognition site, and a HLH domain essential for dimeriza- expression of Cd3d and Cd8 genes within the T-cell lineage, tion. E proteins also share two N-terminal transcriptional activation whereas it directs early B-cell development by the induction of domains, AD1 and AD2, where the AD1 domain modulates Rag gene expression and IgH rearrangements, allelic exclusion of transcription through recruitment of lysine acetyltransferases CBP/ the Igk locus and negative regulation of the Igll1 and Dntt p300 and p/CAF, the Spt-Ada-Gcn5-acetyltransferase (SAGA) promoters, thereby terminating pre-BCR signaling.50 Furthermore, complex and members of the ETO family.72–74 The DNA-binding IKZF1 controls pre-B- by decreasing c-Myc activity of E proteins is abrogated upon binding to members of expression in these cells.59 Beyond the pre-B stage, IKZF1 the class IV HLH proteins comprising ID1–ID4, which contain the controls the function of B cells at later stages of development HLH dimerization motif, but lack a functional DBD. Within the by increasing the activation threshold for mature B cells and regu- hematopoietic compartment, the E2A proteins form heterodimers lating the selection during class-switch recombination.60 with the basic-HLH protein SCL, an essential regulator of Although mouse-knockout models for IKZF1 revealed its hematopoietic development. Proteomic studies have revealed potential role as a tumor suppressor, only with the emergence that E47 also interacts with lysine demethylase LSD1, corepressor of genome-wide screening methods, IKZF1 was identified as a CoREST and members of the 14–3–3 scaffolding proteins.75 bona fide leukemia-suppressor gene in childhood ALL. It has now Furthermore, HEB and E2A bind in a cooperative manner to been established that mono-allelic deletions and point mutations SMAD2 and SMAD3 transcription factors, effectors of TGFb of the IKZF1 gene on chromosome 7p12.2 are a common event in signaling. E12 and E47 are degraded by the ubiquitin- childhood ALL and occur in about 10–15% of BCP-ALL and 4% of proteasome system through binding to ubiquitin-conjugating T-ALL cases2,3 (Figure 4b). These deletions may affect the mUbc9 and via Notch-induced association with E3 complete IKZF1 gene, giving rise to haploinsufficiency, or result ubiquitin SCFSKP2. E2A function is regulated through lysine in intragenic deletions generating DN isoforms due to D 4–6 and acetylation by p300/CBP on residue K34 within the AD1 domain, D4–7 deletions. Furthermore, single-nucleotide polymorphisms at which potentiates transcriptional activation.74 Phosphorylation of the IKZF1 gene correlate with an increased risk to develop ALL.61,62 E12/47 by p38-MAPK interferes with transcription activation,76 Gene deletions and point mutations are also detected in the IKZF3 while phosphorylation on residues S514 and S529 by CKII and gene in about 2% of the BCP-ALL cases.3 Interestingly, IKZF1 protein kinase A inhibits E47 homodimerization.77 In addition, deletions are present at a much higher frequency in high-risk BCR- human E47 harbors an AKT phosphorylation site (S529),75 but the ABL1-positive BCP-ALL (85%),63 while in chronic myeloid leukemia, functional implication remains to be established. IKZF1 deletions are predominantly present in lymphoid blast crises The Tcfe2a/Tcf3 gene is required to maintain the HSC pool (B20%). In line with these observations, Ikzf1 haploinsufficiency under stress conditions and to promote the development of accelerates the onset of B-cell leukemia in p190BCR-ABL1 lymphoid-primed multipotent progenitors, common lymphoid transgenic mice.64 IKZF1 gene deletions also frequently occur in progenitors, early thymocyte progenitors (ETPs), pre-pro-B cells another subclass of high-risk childhood leukemia patients, and all subsequent stages of B-cell differentiation.78,79 A partial characterized by activating JAK2 point mutations that often block at the earliest stages of T-cell differentiation in Tcfe2a À / À harbor high expression of the CRLF2 as a mice precedes the development of T-cell lymphomas in these result of CRLF2 gene rearrangements.65 A large fraction of the mice.80 E2A directs B-cell fate determination by activating Foxo1 remaining IKZF1-mutated BCP-ALL cases also display a BCR-ABL1- gene expression, which, together with E2A, PU.1 and IL-7R- like gene-expression signature, and are often characterized activated STAT5, induce Ebf1 expression. At the same time, E2A by other variants of ABL1 and JAK2 translocations or gene represses myeloid/erythroid-lineage-specifying genes, including lesions in IL7R as identified by transcriptome analyses66 Gata1, Csf1r and EpoR. E47 homodimers are the major E proteins in (Figure 3b). Notably, deletions and mutations of IKZF1 are pro-B lymphocytes, consistent with the specific requirement for associated with a poor clinical outcome and act as a strong E2A proteins at this stage of B-cell differentiation. HEB- and E2-2- predictor of relapse.67,68 deficient lymphocytes develop normally, showing a mild The pathways downstream of IKZF1 that have a critical role in reduction in the number of pro-B cells.81,82 Compound the onset of BCP-ALL and altered therapy response largely remain heterozygous Tcf4 þ / À ;Tcf12 þ / À and Tcf4 þ / À ; Tcfe2a þ / À mice to be identified. In mouse T-cell leukemogenesis, aberrant Notch- show a more severe phenotype compared with the single- receptor activation is a critical key event upon loss of IKZF1 knockout animals, arguing that total level of E proteins, rather function.69 Whether IKZF1-dependent deregulated expression of than the activity of a specific E-box-binding protein, is important Notch or any of its other cancer-related targets, such as CDK- for B-cell development.81 In contrast, E2-2 fulfills a unique and inhibitor p27Kip1 (CDKN1B),70 c-Myc59 or phospho-STAT5,71 essential role in the development of plasmacytoid dendritic contribute to the pathogenesis of BCP-ALL is still an open cells.83 In addition to Ebf1, other E-protein targets important for question. There is compelling evidence that disruption of p19ARF B-lymphoid differentiation include Rag1, Rag2, Dntt, Igll1, Vpreb1 (CDKN2A gene) is a strong cooperating event together with IKZF1 and CD79a, as well as VH–DJH recombination at the IgH locus and gene alterations in Ph þ -ALL, and both represent the predominant Igk rearrangements. Additionally, E2A contributes to activate complementation groups in the BCR-ABL1 subtype of BCP-ALL.3,63 expression of the Pax5 locus in concerted action with EBF1, However, in these leukemia samples, there is still selection for the FOXO1, IRF4 and IRF8.84 In peripheral activated B cells, E47 presence of additional genetic aberrations in other transcription functions to induce Aicda expression and immunoglobulin class- factors in about 60% of the cases, including PAX5, EBF1 and ETV6, switch recombination,85 whereas E2-2 influences follicular versus arguing that mono-allelic IKZF1 gene deletions are not sufficient to marginal-zone fate determination.82 block B-lymphoid differentiation and probably regulate the The human E-protein-encoding genes TCF3, TCF4 and TCF12 additional signaling pathways. are implicated in several different types of hematological

& 2013 Macmillan Publishers Limited Leukemia (2013) 541 – 552 B-lineage transcription factors in leukemia E Tijchon et al 548 malignancies. Recurrent focal gene deletions and point mutations IgH and IgL loci and expression of genes essential for B-lineage affecting TCF4 (chromosome 18q21.1) and TCF12 (chromosome specification and commitment, including Cd79a, Cd79b, Vpreb1, 15q21.3) occur at low frequency in pediatric BCP-ALL.2,4 TCF3 loss- Igll1, Foxo1 and Pax5. EBF1 and PAX5 positively cross-regulate of-function genetic alterations on chromosome 19p13.3 include each other’s promoters at early stages of B-cell development, heterozygous gene deletions, which are found in about 70% of thereby providing an auto-regulatory loop critically important for the patients suffering from Sezary syndrome, a cutaneous subtype B-cell specification. EBF1 antagonizes the expression of Cebpa, of T-cell lymphomas.86 At much lower frequencies, chromosomal Sfpi1, Id2, Id3 and several natural-killer-cell-specific genes.101,102 translocations TCF3-HLF (1%) and TCF3-PBX1 (5%) are found in EBF1 also appears to be a critical regulator of proliferative childhood BCP-ALL (Figure 4c), both of which are associated with expansion and survival of pro-B cells by activating expression of a poor treatment outcome. These oncogenic fusion proteins c-Myb and controlling Akt and canonical B-cell receptor (BCR) combine the transcriptional activation domains of E2A with the signaling.103 Genome-wide chromatin immunopreciptation DBDs of HLF and PBX1, respectively. Both TCF3-HLF and TCF3- analyses have revealed the identification of several new EBF1 PBX1 are thought to interfere with normal E-box-binding/HLH target genes, including Hlx, Ctla4 and Gfi1b.104 Ectopic EBF1 protein function through recruitment of transcriptional coactiva- expression in hematopoietic progenitor cells forces B-cell tors via the AD1 domain of E2A to PBX1 and HLF target genes. development at the expense of myeloid cell fates.101,105 At later Notably, inhibition of E2A/HEB function is an important mechanism stages of B-cell differentiation, EBF1 is essential for the for the frequently occurring gain-of-function alterations in HLH development of B1 and marginal-zone B cells, and maintenance protein-encoding genes TAL1/SCL and LYL1 in T-ALL. In general, of follicular and germinal center B cells.103,106 Ebf1 þ / À mice TCF3-PBX1-positive leukemias display a rather limited amount of display a 50% decrease in pro-B cells and a 10–30% reduction in cooperating gene deletions, including PAX5 (40%) and CDKN2A splenic B cells, but are not prone to develop spontaneous (35%).3 These leukemias are also characterized by almost an leukemia. However, combining a constitutive active transgenic obligatory of the other nontranslocated TCF3 allele (95%).3 STAT5 allele with Ebf1 haploinsufficiency yields progenitor B-cell TCF3-PBX1 is a regulator of BMI1 expression, thereby alleviating leukemias with a penetrance of 100%.107 part of the requirement for CDKN2A gene deletions in BCP-ALL.87 In human ALL, mono-allelic deletions affecting the EBF1 gene TCF3-PBX1 induces the expression of Wnt16, which may affect on chromosome 5q33.3 have been reported in a small fraction of survival signaling and N-cadherin-dependent adhesion.88,89 the total BCP-ALL cohort (4%).3 These deletions are variable in size, Experimental mouse models for TCF3-PBX1-induced leukemia but frequently affect a large portion of the EBF1 open-reading show collaboration with Hox gene overexpression in lymphoid frame, suggesting a loss-of-function phenotype (Figure 4d). malignancies.90 The oncogenic fusion protein TCF3-HLF suppresses Genetic aberrations affecting EBF1 are more prevalent in high- Runx1 transcription and activates expression of Lmo2 and several risk leukemia patients, including BCR-ABL1-positive cases (14%) Groucho-related genes, as well as anti-apoptotic genes Slug and and in a novel defined cluster (38%) characterized by a high Bcl2.91–93 Furthermore, TCF3-HLF mediates overexpression of frequency of TP53/RB1 and JAK mutations, together with other adenosine triphosphate-dependent drug transporter ABCB1 B-cell pathway gene deletions.45 Notably, EBF1 haploinsufficiency (MDR1),94 which may contribute to the poor treatment outcome occurs more frequently in combination with BTG1 gene deletions of leukemias harboring a TCF3-HLF translocation. Thus, TCF3-PBX1 (26%), suggesting that these tumor-suppressor genes may and TCF3-HLF seem to regulate different target genes, but may cooperate in leukemogenesis.108 employ similar mechanisms of ectopic recruitment of transcriptional coactivators, like CBP/p300 and components of the SAGA complex, which are important for the oncogenic PAX5 properties of these leukemia-associated fusion proteins. The guardian of B-cell identity PAX5, also known as B-cell specific activator protein, is one of the nine PAX transcription factors, but the only one expressed within the hematopoietic system. A EBF1 characteristic feature of PAX protein family is the conserved paired The early B-cell factor (EBF) transcription factors EBF1–EBF4 have domain, which functions as a bipartite DNA-binding region and an important role in the development of a number of specialized consist of an N- and C-terminal subdomain. Each subdomain binds cell types, including , adipocytes and lymphocytes. EBF to a distinct half-site of a degenerate consensus recognition proteins are composed of a highly conserved N-terminal DBD, an sequence. PAX5 displays cooperative DNA binding with other IPT/TIG (immunoglobulin, plexins, transcription factors-like/tran- transcription factors, such as MYB and ETS1, and has been shown scription factor immunoglobulin) domain, an atypical helix-loop- to repress PU.1 transactivation through direct binding.109 Besides helix-loop-helix (HLHLH) motif and a less conserved C-terminal the paired domain, PAX5 harbors two interaction domains for domain, which contributes to transcriptional activation. EBF Groucho corepressors,110 a partial homeodomain that serves as a proteins bind DNA as homo- or heterodimers via a unique motif binding domain for pRb,111 and a potent C-terminal transactivation specific for the EBF family, termed the ‘zinc knuckle’ located within domain that interacts with the SAGA complex and lysine the DBD.95,96 EBF1 interacts with transcriptional CBP, acetyltransferases CBP/p300, which induce lysine acetylation of the but surprisingly inhibits its lysine acetyltransferase activity.97 Two PAX5 DBD.112 Alternative splicing of the Pax5 gene generates related large zinc-finger proteins, ZFP423 and ZFP521, can bind multiple protein isoforms that differ in the C-terminal transactivation EBF1 and are capable of recruiting Mi-2b/NuRD complexes. Gene domain, but their individual functions remain to be established. The silencing of Mi-2b subunit seems to potentiate EBF1-mediated death-domain-associated protein DAXX has been shown to bind gene activation.98 Furthermore, EBF1 inhibits DNA methylation of PAX5, thereby modulating its transcriptional activity.113 Proteomic specific target genes (for example, Cd79a), which is counteracted studies have demonstrated that PAX5 can also recruit the TBP, TAF4 by Mi-2b/NuRD. On the other hand, EBF1 requires the BRG1- and and TAF6 subunits of the basal transcription factor complex TFIID, BRM-containing SWI/SNF complex to activate target genes.98 the SWI/SNF chromatin-remodeling components BRG1, BAF57 and The essential role of EBF1 in B-cell lineage commitment has BAF170, the RbBP5 subunit of the MLL-containing H3K4 become evident from various mouse studies. Mice deficient for methyltransferase complex and corepressor NCoR1.114 Thus, PAX5 Ebf1 show an absolute block in differentiation at the pre-pro-B-cell is able to regulate target genes through its interplay with different stage.99 Forced expression of EBF1 in E2A-deficient cells rescues epigenetic modifiers in a context-dependent manner. B-cell differentiation, arguing that EBF1 acts downstream of E2A in During B lymphopoiesis, Pax5 expression starts at the pro-B cell B-cell development.100 EBF1 is essential for the rearrangement of stage acting downstream of E2A and EBF1 in the B-cell

Leukemia (2013) 541 – 552 & 2013 Macmillan Publishers Limited B-lineage transcription factors in leukemia E Tijchon et al 549 differentiation pathway, and its levels are maintained until plasma hematological malignancies. This pathway is activated via ligation cell differentiation occurs. At the early stages of B-cell develop- of secreted Wnt proteins to their receptors and inhibition of the ment, the Pax5 gene is subjected to allele-specific regulation, with Ser/Thr kinase GSK-3b. As a consequence, the transcriptional only one allele being transcribed, whereas it switches to bi-allelic coactivator b-catenin is stabilized, allowing it to translocate to the expression in immature B cells.115 During the initial phase of fetal nucleus and bind to the T-cell factor/lymphoid- factor hematopoiesis, Pax5 deficiency results in a block before the transcription factors TCF7, TCF7L1, TCF7L2 and LEF1. This results in appearance of B220 þ progenitors, while in adult Pax5-deficient a transcriptional switch from repression to activation, through mice, B-cell development proceeds until the c-Kit þ B220 þ pro-B recruitment of coactivator complexes, including BRG1, p300/CBP, cell stage.116,117 Specifically, Pax5-deficient bone marrow cells are the ATAC acetyltransferase complex, BCL9 and the chromatin- 130–133 halted at the stage of VH-to-DJH rearrangement. However, remodeling factors Pygo1/Pygo2. Another level of Pax5 À / À pro-B cells are not yet fully committed to the B-cell complexity is introduced by the expression of distinct isoforms lineage, because these cells remain pluripotent and are able to for each of the T-cell factor/lymphoid-enhancer factor family differentiate into functional macrophages, granulocytes, members, where alternative splicing generates four different LEF1 dendritic cells, osteoclasts and natural killer cells in the presence isoforms. Moreover, there is a natural occurring LEF1-antisense of the appropriate .118 Enforced expression of PAX5 in transcript located at the 50 end of the gene, which has not yet multipotent progenitors results in the production of biphenotypic been studied in great detail. LEF1 contains a DBD that shows myeloid/B-lymphoid cells with extensive self-renewal capacity.119 homology to members of the high-mobility group proteins, and PAX5 fulfills a dual role as master regulator of lineage binds DNA as a monomer inducing a sharp bend in the DNA. In commitment by repressing non-B-lineage genes (Notch1, Csf1r, the absence of Wnt signaling, LEF1 predominantly acts as a Csf1 and CD28) and differentiation-stage inappropriate genes (Flt3, transcriptional repressor by assembling CtBP, TLE/Groucho and Sca1 and Blimp1), while activating (pro-) B-cell-specific genes, HDAC molecules to its target genes.133 including components of receptor signaling (Cd79a, Cd19, Blnk, The Wnt/b-catenin signaling pathway controls the proliferation, Cd72 and Igk) and genes encoding transcriptional regulators (Ebf1, survival and differentiation of hematopoietic cells. Self-renewal of Lef1, Tcf4, Ikzf3, Id3, Bach2, Irf4 and Irf8). In addition, PAX5 permits HSCs coincides with activation of the b-catenin pathway, and high accessibility of the immunoglobulin heavy chain locus through levels of constitutively activated b-catenin in HSCs confer myeloid binding to PAX5-activated intergenic repeat elements.120 PAX5 potential on lymphoid progenitors.134 Mice deficient for Lef1 display expression is enhanced in activated B cells and essential for class- reduced proliferation and enhanced apoptosis of pro-B cells, but no switch recombination, by activating Aid expression. However, defect in B-cell differentiation,135 whereas ectopic LEF1 expression PAX5-target genes in early and late B lymphopoiesis appear to be promotes myeloid at the expense of lymphoid development.136 quite distinct.121 PAX5 remains required for maintaining the B-cell Moreover, enforced LEF1 expression in bone marrow progenitor gene-expression program, as conditional Pax5 gene inactivation cells results in myeloid and lymphoid malignancies upon results in the conversion of mature B cells into functional T cells by transplantation into lethally irradiated recipient mice.136 In human dedifferentiation to uncommitted progenitors in the bone CD34 þ cells, inhibition of LEF1, but not of b-catenin, affects marrow.122,123 In addition, complete loss of PAX5 in mature B proliferation and apoptosis of these cells, suggesting that LEF1 may cells leads to the development of aggressive progenitor B-cell fulfill b-catenin-independent functions in early human progenitor lymphomas in these mice.123 In vivo studies have shown that cells.137 Furthermore, these studies have demonstrated a role of constitutive active STAT5 expression in combination with Pax5 LEF1 in differentiation, which correlates with regulation haploinsufficiency drive ALL development.107 On the other hand, of the ELA2 gene by LEF1 together with CBFa. ectopic PAX5 expression in the T-cell lineage induces immature Enhanced activation and strong nuclear localization of lymphoblastic T-cell lymphomas in IgH-PAX5 transgenic mice. b-catenin has been demonstrated in chronic myeloid leukemia, The PAX5 gene located on chromosome 9p13.2 is deregulated ALL, AML, mantle cell and cutaneous lymphoma. The one way this in a small fraction of diffuse large B-cell lymphomas, which results pathway can be modulated is through epigenetic from translocations involving the IgH enhancer (Em) and correlate of Wnt antagonists, as observed in AML and ALL.138,139 In addition, with elevated PAX5 levels.124 In addition, aberrant ectopic overexpression of LEF1 has been observed in CLL, ALL, AML and expression of PAX5 has been reported in AML.125 On the other lymphomas and associated with enhanced survival properties of hand, PAX5 loss-of-function genetic lesions have been identified in CLL cells and poor treatment outcome in adult BCP-ALL a high percentage of childhood and adult BCP-ALL cases (±35%), patients.140,141 On the other hand, recurrent inactivating mono- which include predominantly mono-allelic deletions that allelic and bi-allelic microdeletions, and truncating point sometimes involves a larger segment of chromosome 9p and mutations of the LEF1 gene, located on chromosome 4q25, have point mutations, as well as some chromosomal translocations been reported in pediatric T-ALL (17%) and BCP-ALL (2%).3,142 generating a large variety of distinct chimeric PAX5 fusion Somatic mutations in TCF7L2 have also been described in BCP- proteins (Figure 4e). PAX5 mutations have also been identified in ALL.143 Deficiency for LEF1 function has been linked to enhanced splenic marginal-zone lymphomas and diffuse large B-cell c-MYC and FAS expression,135,142 but whether these LEF1-target lymphomas.126 Recurrent translocations in BCP-ALL include genes critically contribute to human leukemogenesis remains to PAX5-ETV6, PAX5-C20orf112, PAX5-FOXP1 and PAX5-PML that be determined. predominantly act to suppress PAX5-target genes.3,127 PAX5 CNAs are detected in virtually all BCP-ALL genetic subtypes, but occur more frequently in BCR-ABL1- (50%) and TCF3-PBX1-positive CONCLUDING REMARKS cases (41%).3,128 The frequent concurrent deletion of PAX5 and Genomic profiling by single-nucleotide polymorphism arrays and CDKN2A suggests that they are cooperating events in the genomic sequencing have led to the identification and character- pathogenesis of BCP-ALL.3,129 There is no significant association ization of novel genetic lesions that not only contribute to the between clinical outcome and PAX5 status, despite the finding pathogenesis of BCP-ALL, but in some cases also predict therapy that PAX5 is commonly mutated in ALL.67 response. It is evident that the B-lineage transcription factors described in this review have a central role in the differentiation and survival of lymphocyte progenitor cells. However, for many of LEF1 these factors, it remains to be established how they contribute to Deregulation of the canonical Wnt/b-catenin signaling pathway leukemia development or which cooperating events are required has been identified as a hallmark of multiple , including to develop the disease. In addition, the observation that both

& 2013 Macmillan Publishers Limited Leukemia (2013) 541 – 552 B-lineage transcription factors in leukemia E Tijchon et al 550 gain-of-function and loss-of-function genetic lesions that affect 16 Zhao X, Vu LP, Perna F, Liu F, Xu H, Nimer S. Methylation of RUNX1 by the proteins, such as AML1, ERG, PAX5 and LEF1, may contribute the nuclear PRMT5/COPR5 complex regulates its cellular location in Leukemia cells. oncogenic transformation of hematopoietic cells, calls for a more Blood (ASH Annual Meeting Abstracts) 2011; 118: 3403. detailed understanding of the distinct signals regulated by these 17 Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target transcription factors. Extensive network structure analyses will of multiple chromosomal translocations in human leukemia, is essential for provide additional insight into the key target genes that are normal fetal liver hematopoiesis. Cell 1996; 84: 321–330. regulated by these mutated transcription factors.144 The next step 18 Sasaki K, Yagi H, Bronson RT, Tominaga K, Matsunashi T, Deguchi K et al. Absence will be to translate this knowledge into improved therapy of fetal liver hematopoiesis in mice deficient in transcriptional coactivator beta. Proc Natl Acad Sci USA 1996; 93: 12359–12363. modalities for the patient. These may include drugs that 19 Growney JD, Shigematsu H, Li Z, Lee BH, Adelsperger J, Rowan R et al. Loss of interfere with the action of epigenetic regulators recruited by Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative oncogenic fusion proteins, such as ETV6-RUNX1 and TCF3-PBX1. It phenotype. Blood 2005; 106: 494–504. can be expected that such developments will lead the way from 20 Motoda L, Osato M, Yamashita N, Jacob B, Chen LQ, Yanagida M et al. Runx1 standard (chemo-)therapy to personalized medicine for patients protects hematopoietic stem/progenitor cells from oncogenic insult. Stem Cells with BCP-ALL. 2007; 25: 2976–2986. 21 Seo W, Ikawa T, Kawamoto H, Taniuchi I. Runx1-Cbfbeta facilitates early B lympho- cyte development by regulating expression of Ebf1. JExpMed2012; 209: 1255–1262. CONFLICT OF INTEREST 22 Blyth K, Slater N, Hanlon L, Bell M, Mackay N, Stewart M et al. Runx1 promotes The authors declare no conflict of interest. B-cell survival and lymphoma development. Blood Cells Mol Dis 2009; 43: 12–19. 23 Kundu M, Compton S, Garrett-Beal L, Stacy T, Starost MF, Eckhaus M et al. Runx1 deficiency predisposes mice to T-lymphoblastic lymphoma. Blood 2005; 106: 3621–3624. ACKNOWLEDGEMENTS 24 Putz G, Rosner A, Nuesslein I, Schmitz N, Buchholz F. AML1 deletion in adult mice We thank RP Kuiper, LT van der Meer and C Murre for critically reading the causes splenomegaly and lymphomas. 2006; 25: 929–939. manuscript. 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