The Pre-B-Cell Receptor Checkpoint in Acute Lymphoblastic Leukaemia

REVIEW The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia

J Eswaran1, P Sinclair1, O Heidenreich1, J Irving1, LJ Russell1, A Hall1, DP Calado2,3, CJ Harrison1 and J Vormoor1,4

The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

Leukemia (2015) 29, 1623–1631; doi:10.1038/leu.2015.113

INTRODUCTION surface protein receptors (Figure 1).11,12 Initially, multipotent Acute lymphoblastic leukaemia (ALL) results from expansion of lymphoid progenitors, generated from haemopoietic stem cells, immature haematopoietic cells in the bone marrow and blood. It differentiate into early pro-B cells. Subsequent rearrangements of is the most common childhood malignancy, with a peak incidence the Ig heavy-chain (HC) variable (V), diversity (D) and joining (J) around 2–5 years of age. In children, the frequency is 3–4 cases gene segments give rise to expression of an IgM HC that interacts per 100 000 each year, while among adults, the annual incidence with non-polymorphic ‘surrogate’ light-chain (LC) components to 13,14 is lower, around 1 case per 100 000. Treatment modifications, form the pre-B-cell receptor (pre-BCR). In human BCP-ALL, the improved patient management and risk stratification have B-cell developmental process is arrested at this pre-B-cell stage dramatically improved survival rates to more than 80% for (Figure 1). In support of these observations, when expression of children and about 40% for adults.1 Acquired chromosomal pre-BCR was hampered through deletion of the exons coding for abnormalities are the hallmark of ALL, which define biologically the transmembrane region, IgHM (Immunoglobulin Heavy distinct subtypes of the disease. The strong association between Constant Mu), in μMT mice, it resulted in a complete arrest of cytogenetic subtypes and prognosis has further refined risk B-cell development with twofold enrichment of pro-B cells.15 stratification for treatment in a large number of protocols The pre-B-cell checkpoint is one of the first vital cell fate worldwide.2–4 Research into the biological and clinical roles of decision-making elements that controls critical B-cell develop- additional genetic alterations, including copy number abnormal- mental processes, such as clonal selection, expansion and 13,16 ities and gene mutations within key pathways, such as B-cell subsequent maturation to the pre-B-cell stage (Figure 1). differentiation and development, cell cycle control, RAS Once the pre-BCR complex is assembled, its transient expression (Rat sarcoma viral oncogene homologue) and cytokine signalling, induces signalling that initiates clonal expansion of IgHM positive 17 are ongoing.5–7 pre-B cells. When a number of cell proliferation cycles have been ALL is a clonal disease, originating from precursor B- or completed, pre-BCR signalling arrests the cell cycle, which is a T-lineage cells,1 which become the propagators of the leukaemia. pre-requisite for entry into small pre-B cell stage and further Almost all blasts have leukaemia-propagating potential in BCP- cell maturation. More importantly, B lymphocytes with a ALL,8,9 forming a model completely different from the hierarchical non-productive Ig HC are eliminated through programmed cell model of acute myeloid leukaemia and normal haemopoietic stem death. Thus, the function of pre-BCR checkpoint and its down- cells.8,10 These observations highlight the need to understand how stream signalling is to select pre-B cells that are suitable for BCP-ALL cells manage to evade early B-cell developmental continuing development or otherwise initiate cell death. After the checkpoints during the development of leukaemia. pre-BCR checkpoint positive selection, the occurrence of LC B-cell development is a highly complex process involving rearrangement leads to the next developmental stage, the rearrangement of the immunoglobulin (Ig) genes, as well as cell immature naive B cells. These naive immature B cells are positively

1Leukaemia Research Cytogenetics Group, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK; 2Cancer Research UK, London Research Institute, London, UK; 3Peter Gorer Department of Immunobiology, Kings College London, London, UK and 4Great North Children’s Hospital, Newcastle-upon-Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. Correspondence: Professor CJ Harrison, Leukaemia Research Cytogenetics Group, Northern Institute for Cancer Research, Newcastle University, Level 5, Sir James Spence Institute, Royal Victoria Inﬁrmary, Newcastle upon Tyne NE1 4LP, UK. E-mail: [email protected] Received 18 November 2014; revised 20 April 2015; accepted 23 April 2015; accepted article preview online 6 May 2015; advance online publication, 22 May 2015 pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1624

Figure 1. Normal B-cell development from the pro-B cell to immature B-cell stage in the bone marrow, showing how the blockade in pre-BCR expression, the pre-BCR regulated processes, such as cell cycle control, clonal extinction and apoptosis, lead to the development of BCP-ALL.

selected for expression of a non-auto reactive, functional BCR and 3. Bypassing the pre-BCR checkpoint and activating pro-survival leave the bone marrow and differentiate into distinct B-cell signalling through pre-BCR independent alternative mechanisms. subtypes such as follicular B cells, marginal zone B cells and germinal centre B cells.13,18 The activation and clonal expansion of We review the application of these mechanisms in BCP-ALL these cells occur upon antigen-induced BCR signalling as well as and discuss pre-BCR-mediated BCP-ALL signalling pathways as signalling from cytokine and co-stimulatory receptors. Thus, the potential therapeutic targets. BCR structure, expression and signalling status (that is, tonic or chronic active) function as a ‘homoeostatic controller’ of B-cell development.13,19 BLOCKING PRE-BCR EXPRESSION AND FUNCTIONS FAVOUR Throughout lymphoid development, B-lineage cells undergo BCP-ALL DEVELOPMENT stringent selection for expression of pre-BCR and the appropriate Arrest in differentiation at the pre-BCR checkpoint is specificto BCR. Although expression of pre-BCR is blocked in the majority of BCP-ALL. Therefore, a key challenge is to understand why BCP-ALL, most mature B-cell malignancies express BCR and the differentiation is blocked at the pre-B-cell stage. A most recent development of the tumour is often driven by the intensity of study investigated the expression of pre-BCR components (IGLL1, – skewed BCR signalling.13,18 20 Using different BCR signalling IGLL3, VPREB1, VPREB3 and IGHM) and the presence of functional intensities, for example, (i) absent,21 (ii) basal/tonic22 and tonic pre-BCR signalling in 830 cases of BCP-ALL (including MLL, (iii) chronic active,21,23 and the regulators of BCR-mediated survival BCR-ABL1, ETV6-RUNX1, B-other, PBX1 rearrangement, 6q21 dele- pathways, mature B-cell malignancies are classified, which has led tion, duplication of 1q23 and high hyperdiploid ALL), and found to the development of new treatment strategies.19,24 In most only 112 (13%) to be positive for pre-BCR expression.20 Given the B-cell lymphomas, the tumour progression depends on BCR fact that pre-BCR induces pro-proliferative activity, a process expression, with few exceptions, namely classical Hodgkin’s which in itself would be assumed to promote the development lymphoma, primary mediastinal B-cell lymphoma, some post- of BCP-ALL, it is intriguing that the vast majority of the BCP-ALL transplant lymphomas, and rare primary effusion lymphomas.25,26 lack pre-BCR expression/function. It is possible that pre-BCR may In several lymphomas, BCRs arrested at a particular stage of function as a tumour suppressor under certain leukaemic differentiation, for example, tonic, auto reactive or antigen bound conditions. Such a role has been demonstrated in Philadelphia state, contribute to lymphoma pathogenesis. The role of BCR chromosome (Ph)/BCR-ABL1 (breakpoint cluster region protein— signalling pathways in B-cell development and malignancies have Abelson murine leukaemia viral oncogene homologue 1)-positive been extensively reviewed.13,18,19,27 In contrast, the status of pre- ALL. The leukaemic pre-B cells evade normal B-cell differentiation BCR in ALL and its influence on the downstream signalling in and survive by avoidance of the pre-BCR checkpoint, likely due to BCP-ALL has been the rarely studied; therefore this review is the selection of non-functional IgHM gene rearrangements.28 The focussed on the role of pre-BCR-mediated signalling in BCL-ALL. tumour suppressor function of the pre-BCR has been clearly Here, we present evidence to show that malignant blasts avoid demonstrated by re-expression of a functional μ-chain in primary clonal extinction by hijacking pre-BCR signalling pathways ALL cells with non-functional IgHM rearrangements.29 The (Figure 2) in favour of the development of BCP-ALL. The different successful reconstitution of pre-BCR signalling has been shown mechanisms include the following: to effectively suppress leukaemic growth in Ph-positive ALL.30 In further support, BCR-ABL1 has been shown to downregulate the 1. Blocking the expression of the pre-BCR complex to evade expression of the pre-BCR genes, VPREB1 (VpreB protein) and normal clonal selection and cell cycle arrest. IgHM, linking non-functional IgHM rearrangements, as seen in 2. Activating pre-BCR-mediated pro-survival and pro-proliferative Ph-positive ALL, with a non-functional pre-BCR.30 In addition, signalling, while inhibiting cell cycle arrest and maturation. BCR-ABL1 has also been shown to result in truncated isoforms of

Figure 2. The signalling pathways regulated by pre-BCR in normal B-cell development. Pre-BCR signalling is induced by auto-crosslinking of the surrogate light chains, which triggers the phosphorylation of ITAM (immunoreceptor tyrosine-based activation motif) tyrosine residues of Igα and Igβ by SRC family kinases such as SYK (spleen tyrosine kinase), ZAP70 (ζ-chain-associated protein kinase of 70 kDa), BTK (Bruton tyrosine kinase) and Lck/Lyn (Yes-Related Novel Protein Tyrosine Kinase). When ITAM is phosphorylated by SYK and BTK due to PI3K activation, the B-cell linker protein, BLNK, is recruited and phosphorylated. Subsequently, BLNK induces the RAS–extracellular signal-regulated kinase (ERK) pathway that positively regulates cell cycle. For the negative regulation of cell cycle, phosphorylated BLNK inhibits the JAK3-STAT pathway, AKT activation, which consequently arrests PAX5 activation while promoting FOXO1 (forkhead box protein O1) nuclear localisation. Cell proliferation signalling is also induced by the PI3K-JAK3-STAT pathway, as well as phosphorylation of the apoptotic regulator, BACH2. The target genes induced or repressed downstream of pre-BCR signalling through various transcription factors are shown in italics. The pre-BCR signalling components that function as, tumour suppressors (green) and reported to be constitutively active (red) or inactivated through deletion, mutation and splicing (blue) in BCP-ALL are shown using asterisks.

SLP65, which prevents expression and function of the pre-BCR. illustrated how negative selection mechanisms that enable The treatment of Ph-positive ALL primary leukaemic cells with elimination of such non-functional Ig HCs of pre-BCR are modiﬁed imatinib, a speciﬁc inhibitor of BCR-ABL1 kinase activity, remove to facilitate the development of BCP-ALL.32 The transcriptional this truncated isoform, enabling the blasts to differentiation into repressor, BACH2, is activated by PAX5 (Paired Box 5), while its immature B cells expressing Ig LC.29 A recent report of focal phosphorylation by the PI3K (phosphatidylinositide 3-kinases)/ deletions within VPREB1 in BCP-ALL noted a high frequency of ribosomal s6 kinase pathway maintains the cytoplasmic localisa- VPREB1 deletions in a wide range of ALL subtypes, further tion of BACH2, preventing transcriptional activity (Figure 2). corroborating a tumour suppressor role.31 In early pre-B cells, the absence of a functional pre-BCR triggers high expression of BACH2, which in turn activates TP53 (tumour protein p53), eliminating pre-B cells without IgHM EVASION OF CLONAL SELECTION AND CELL CYCLE ARREST recombination.33 In contrast, when signals are transduced from When malignant clones evade the pre-BCR checkpoint, they competent pre-BCR, BCL6 expression is activated. This process encounter clonal extinction mechanisms that eliminate the non- consequently results in suppression of CDKN2A (Cyclin-Dependent functional Ig HC of pre-BCR during normal B-cell development. Kinase Inhibitor 2A or p14Arf) and TP53, leading to cell survival Recent studies of the transcription factors, BACH2 (bric-a`-brac, and proliferation. Interestingly, BACH2 and BCL6 compete for tramtrack and broad complex and cap’n’collar homology) and binding to the promoter regions of CDKN2A and TP53 in normal as BCL6 (B-cell chronic lymphocytic leukaemia/Lymphoma 6), have well as malignant B-cell precursors. Consequently, the equilibrium

© 2015 Macmillan Publishers Limited Leukemia (2015) 1623 – 1631 pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1626 between BACH2 and BCL6 determines whether transcriptional activation and retention of FOXO1 (Forkhead box O1) proteins activation or repression of CDKN2A and TP53 is triggered that target the transcriptional regulator, BCL6 (Figure 2). FOXO1 (Figure 3). inhibits transcription of CCND2 (Cyclin D2) and MYC, which, by As a result of these critical clonal selection functions mediated acting as tumour suppressors, results in termination of prolifera- by pre-BCR, the expression of BACH2 is frequently downregulated tive expansion. In accordance with these observations, the 32 in BCP-ALL. Notably, low levels of BACH2 expression have been conditional inactivation of FOXO1 at the pro-B-cell stage leads associated with relapse in BCP-ALL. Additionally, a tumour to a complete block in B-cell development.42 Similar findings have suppressor function of BACH2 has been reported among been reported in mice, where the tumour suppressor function of the 30% of BCP-ALL with PAX5 deletions, translocations or 6,34,35 Slp65 appears to be mediated through inhibition of JAK3, by direct mutations. In most PAX5 fusions, including PAX5-ETV6 (Ets interaction as well as indirect mechanisms,43 and by induction of Variant 6) and PAX5-C20orf112 (chromosome 20 open reading BCL6-mediated cell cycle arrest via downregulation of Myc.44 frame 112), the N-terminal DNA binding domain of PAX5 is In addition, SLP65 signalling controls the expression of several retained, while the C-terminal PAX5 activation domain is removed. transcription factors, such as SPIB (Spi-B transcription factor), IRF4 This modification allows the fusion protein to bind to the PAX5 (interferon regulatory factor 4) and IKZF3 (also known as the Ikaros DNA motif and inhibit the function of endogenous PAX5 in a fi κ dominant-negative fashion, which consequently affects the family zinc nger protein, Aiolos), which regulate Ig gene expression of target genes involved in B-cell differentiation, recombination within the nucleus. The central role of SLP65 in including BACH2.34,36 mediating cell cycle arrest and maturation and how leukaemic In a similar manner, suppression of CDK2NA/Mdm2/p53 cells attempt to avoid this checkpoint is apparent in BCR-ABL1 mediated cell death occurs when the tumour suppressor, SLP65 expressing B-lymphoid blasts with a specific intragenic deletion of (SH2 domain containing leukocyte protein of 65kDa also known as the transcription factor, IKZF1 exons 4–7, or loss of the N-terminal BLNK and BASH), is deleted in Slp65−/− leukaemic mice.37 Slp65 zinc finger region that mediates DNA binding. These deletions knockout mice have a 5–10% incidence of pre-B-cell leukaemia, produce the dominant-negative IKZF1 isoform, Ik6.45 The expres- while re-expression of SLP65 prevents leukaemia formation.38,39 sion of Ik6 leads to resistance to pre-BCR induced cell cycle arrest. SLP65 is a key cell cycle regulator that switches cell fate from However, re-expression of full-length IKZF1 leads to tyrosine proliferation to differentiation, facilitating the transition from large phosphorylation of SLP65 at Y96, which inhibits JAK-STAT (Janus 39 cycling into small resting pre-B cells. Downstream of pre-BCR kinase-signal transducer and activator of transcription) signalling signalling, the non-receptor tyrosine kinases, SYK (spleen tyrosine and promotion of cell cycle arrest.30 kinase) and BTK (Bruton agammaglobulinemia tyrosine kinase), In keeping with these tumour suppressor functions of SLP65, phosphorylate SLP65 and enable it to function as a docking loss of SLP65 function in BCP-ALL, resulting from the insertion of molecule for several cytosolic proteins (Figure 2). Through these alternative exons into SLP65 transcripts, has been reported to lead interactions, active SLP65 regulates the cell cycle in either a to premature stop codons.38 However, when the expression of positive or negative manner through different downstream SLP65 was studied in 309 BCP-ALL patients, it was shown to have pathways. a similar level to the normal counterparts in the majority of cases46 In negative feedback regulation of the cell cycle, the SLP65-BTK indicating that loss of expression as a mechanism to avoid SLP65- dependent tumour suppressor pathway downregulates the mediated tumour suppression is rare in BCP-ALL. A number of expression of SLC (surrogate light chain) and IL-7R (Interleukin-7 receptor α-chain),11,39,40 which terminates cell proliferation and studies have provided evidence of synergistic tumour suppressor induces Igκ recombination through various downstream functions of BTK and SLP65 in BCP-ALL: (i) in infant BCP-ALL, 39,41 reduced BTK expression and production of defective BTK pathways. Phosphorylated SLP65 reduces AKT (V-Akt Murine 47 Thymoma Viral Oncogene homologue) activity, leading to the transcripts are linked to resistance to apoptosis, (ii) the Btk/SLP65 double mutant mice shows an increased incidence of pre-B-cell tumours (75% at 16 weeks of age) compared with SLP65 deficient mice and (iii) a low-level expression of the constitutively active form of Btk (E41K-Y223F mutant), prevents tumour formation in Btk/SLP65 double mutant mice.41 Interestingly, a recent report of X-Linked agammaglobulinemia with BCP-ALL showed substitution of an amino acid at codon 307 of BTK indicating that BTK may have a role in BCP-ALL initiation.48,49 In addition to the SLP65-BTK tumour signalling axis, many other novel mechanisms steer the key regulatory molecules involved in cell cycle arrest towards malignant transformation at the pre-BCR checkpoint in BCP-ALL. Deletions within the tumour suppressor, CDKN2A, are frequent secondary events in both BCP- and T-ALL.50 CDKN2A encodes two genes: p16INKa, an inhibitor of the tumour suppressor, Rb (Retinoblastoma), and the cyclin-dependent kinase and p14ARF, an inhibitor of MDM2-mediated ubiquitination. Deletion of CDKN2A in BCP-ALL results in degradation of the p53 pathway, which controls the tumour suppressor activities of both Rb and p53 leading to aberrant self-renewal of the leukaemia cells. Moreover, homozygous deletions of CDKN2A have also been suggested to have a specific role in leukaemic transformation of dic(9;20)(p13·2;q11·2)-positive BCP-ALL.51 Taken together, these Figure 3. The key components involved in one of the major pre-BCR data suggest that pre-BCR functions as a novel tumour suppressor, signalling-associated cell cycle regulation switch, BACH2-BCL6 and cell cycle regulator and a central antigen-independent switch that how the balance between them determines whether to execute performs the first ‘quality control’ check in the B-cell differentia- apoptosis or survival. tion programme.

Leukemia (2015) 1623 – 1631 © 2015 Macmillan Publishers Limited pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1627 ACTIVATION OF PRE-BCR-MEDIATED SURVIVAL AND blocking several downstream signalling molecules involved in cell PRO-PROLIFERATIVE SIGNALLING IN BCP-ALL cycle control.63 This mechanism is supported by the observations The induction of pre-BCR proliferative signalling occurs when the that, when pre-BCR components were reconstituted in receptor- surface non-immunoglobin component of pre-BCR, the lambda 5 deficient pre-B cells, PI3K, AKT and FOXO proteins were shown to 42,64,65 protein, interacts and aggregates on the cell surface.52 It has also be regulated by pre-BCR signalling through SYK. been suggested that stromal proteins, such as galactin 1 and One of the best examples of the effect of pre-BCR signalling on 53,54 proliferation in BCP-ALL is in Ph-positive ALL, in which BCR-ABL1 heparin, interact with pre-BCR to induce pre-BCR signalling. 66 Many immune receptors, including pre-BCR, have been shown to mimics constitutively active pre-BCR signalling. BCR-ABL1 leads induce downstream signalling independent of antigen binding, a to abnormal splicing of BTK mRNA. A truncated isoform of BTK, process referred to as ‘tonic signalling’.13,55 Interestingly, there are which lack kinase activity, BTK52, is able to form a link between functional similarities reported between auto reactive BCR and BCR-ABL1 and the full-length BTK protein, via its SRC-homology pre-BCR signalling.56 The pre-BCR employs an unusual mechanism domain, leading to its phosphorylation and activation in the that allows surrogate LC binding through N-linked glycosylation at absence of a functional pre-BCR. This process leads to phosphor- position asparagine 46 of μHC of pre-BCR.57 This post-translational ylation of LYN, HCK (Hematopoietic Cell Kinase) and FGR (Feline modification is shown to be critical for the interaction between Gardner-Rasheed Sarcoma Viral Oncogene Homologue or Src2) and CBL (Cbl Proto-Oncogene), promoting constitutive survival surrogate LC and HC, which is a pre-requisite for the HC and LC 30 association and successful assembly of pre-BCR. and proliferation. Once pre-BCR is assembled, one of the first steps to occur at the The functions of another key pro-survival molecule, BCL6, are membrane is recruitment of the Src kinase, LYN (Lck/Yes-Related also manipulated in BCP-ALL. In normal pre-B cells, BCL6 is the key Novel Protein Tyrosine Kinase) and the cytoplasmic tyrosine downstream survival factor that mediates clonal rescue following the expression of a functional IgM and cell surface assembly of the kinase, SYK. They phosphorylate the tyrosine residues in 67 immunoreceptor tyrosine-based activation motifs in the cytoplas- pre-BCR. BCL6 also executes its survival signal by binding to the TP53 promoter, suppressing TP53 transcription.68 A recent study in mic tails of Igα and Igβ. When SYK binds to phosphorylated adult MLL-rearranged BCP-ALL demonstrated that MLL-rearrange- immunoreceptor tyrosine-based activation motif, it induces ments are associated with hypomethylation and overexpression of autophoshorylation as well as phosphorylation of SYK by other the BCL6 gene.69 BCL6 is also upregulated in Ph-positive ALL in tyrosine kinases. This step promotes phosphorylation of the pre- response to exposure to tyrosine kinase inhibitors, mediating BCR immunoreceptor tyrosine-based activation motif in a positive tyrosine kinase inhibitor resistance and cell survival.68 In addition feedback loop. to the downstream cell survival and proliferation signalling Although pre-BCR itself has no tyrosine kinases attached to the molecules, knockdown of the Igα or Igβ chains of the pre-BCR cytoplasmic segment, SYK kinase has a central role in pro-B- to complex has been shown to reduce viability of TCF3-PBX1 pre-B-cell differentiation. The active SYK phosphorylates SLP65, (Transcription factor 3-Pre-B-Cell leukaemia transcription factor and SYK-SLP65-mediated positive cell cycle regulation occurs 70 γ γ 1)-positive ALL cell lines. Similarly, treatment of ibrutinib, a BTK when phosphorylated SLP65 binds to PLC 2 (phospholipase C 2). inhibitor, also significantly reduced the cell viability of all TCF3 This action in turn hydrolyses phosphatidylinositol 4,5-bispho- rearranged cases that intrinsically expressed higher protein levels sphate to generate diacylglycerol and inositol1,4,5-trisphosphate, fl 58 γ of pre-BCR pathway molecules such as SYK, LYN, SLP65, BTK and inducing calcium in ux. Moreover, PLC 2 indirectly activates PLCγ2.71 Thus, pre-BCR expression, downstream cell proliferation RAS, promoting the activation of ERK (extracellular signal- and cell cycle control signalling are skewed in favour of BCP-ALL regulated kinase) which induces cell proliferation. Within the development in different subtypes of BCP-ALL. RAS-ERK pathway, ERK1 and ERK2 are mediated by the ERK transcription factor targets, Elk1 (ETS-like gene 1) and CREB (cyclic AMP Response Element-Binding Protein), which target Myc (v-myc BYPASSING THE PRE-BCR CHECKPOINT: PRE-BCR- avian myelocytomatosis viral oncogene homologue) or Mef2c INDEPENDENT SURVIVAL AND PRO-PROLIFERATIVE (monocyte enhancer factor 2c) and Mef2d, respectively SIGNALLING IN BCP-ALL 59,60 (Figure 2). In addition to pre-BCR-mediated BCP-ALL signalling pathways, In addition, ectopic expression of SYK leads to constitutive there are several alternative unconventional pre-BCR-independent activity that enables SYK to function as an oncoprotein. Due to its mechanisms known to trigger malignant transformation in BCP- role in promotion of proliferation and inhibition of apoptosis in ALL. For example, when BCP-ALL cells were stimulated in vitro pre-B cells, the exogenous overexpression of SYK transforms using IL7, PI3K was activated, which in turn activated the JAK-STAT normal pre-B cells. Similarly, endogenous SYK activity has been pathway, resulting in increased survival and proliferation of these shown to be essential for MYC induced pre-B cell malignant cells.64 In TCF3-PBX1-positive ALL cells, inhibition of pre-BCR 61 transformation. Inhibition of SYK activity promotes apoptosis of signalling by Dasatinib and knockdown of Igα and Igβ, leads to primary human BCP-ALL in vitro and in vivo, validating SYK as a upregulation of ROR1 (receptor tyrosine kinase-like orphan 61,62 drug target for BCP-ALL. receptor 1).70 ROR1 is an embryonic protein whose expression is In normal pre-B cells, immunoreceptor tyrosine-based activation found in hematogones, which are non-neoplastic B-lymphocyte motif phosphorylation and activation of SYK triggers various precursor-like cells found in the bone marrow of children.72 downstream cell proliferation signalling pathways (Figure 2). The Although the role of ROR1 in normal B-cell precursors remains active SYK phosphorylates and activates PI3K (phosphoinositide 3 to be determined, silencing of pre-BCR expression induces kinase) directly, as well as indirectly through phosphorylation of upregulation of ROR1, suggesting an intrinsic connection between CD19 and the adaptor protein, BCAP (B-cell PI3K adaptor). the two.72 The ROR1 immunoprecipitation studies of TCF3-PBX1- Subsequently, PI3K phosphorylates its substrate phosphatidylino- positive BCP-ALL samples showed RAS GTPase-activating proteins sitol‑4,5‑bisphosphate, which leads to production of the second to function downstream of ROR1. When phosphorylation levels of messenger, PtdIns(3,4,5)P3 (phosphatidyl‑inositol‑3,4,5‑trispho- MEK and ERK were measured after ROR1 knockdown in the same sphate) and recruitment of other kinases, such as AKT and PDK1 subtype, they were remarkably decreased. In the absence of pre- (3-phosphoinositide‑dependent protein kinase1) through their BCR and its downstream AKT cell viability pathway, upregulation pleckstrin‑homology domain to the membrane. This pre-BCR of ROR1 was shown to activate the pro-survival ROR1/MEK/ERK signalling pathway suppresses expression of RAG1, which is pathway.70 These novel BCP-ALL-specific signalling networks required for differentiation, and induces cell proliferation through provide survival signals independent of pre-BCR.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1623 – 1631 pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1628 Another mechanism indirectly impacting on pre-BCR function In addition to pre-BCR, other membrane co-stimulatory was observed in ETV6-RUNX1-positive ALL, associated with regulators that mediate cell-survival pathways have been proven aberrant expression of EPOR (erythropoietin receptor).73–75 This to offer new therapeutic opportunities. For example, CD19, the overexpression occurs through different mechanisms, including type I membrane glycoprotein antigen, is highly expressed in binding of ETV6-RUNX1 fusion proteins to the EPOR promoter74,76 more than 90% of BCP-ALL.90 It positively regulates the expression and rearrangement with the IGH (immunoglobulin heavy-chain of IL-7R in B-cell progenitors.91 In pre-BCR positive BCP-ALL cases, locus) enhancer. The IGH translocation, t(14;19)(q23;p13),77 and CD19 may have a significant role by subverting the PI3K/AKT cell insertion of EPOR into IGH78 lead to overexpression of EPOR, in the survival pathway.90 A recent study demonstrated that CD19 same manner as other partner genes controlled by IGH. The synergistically functions with the TNF receptor family member, functional consequence of this translocation in lymphoid cells is BAFFR, enabling pro-survival of mature B cells under SYK null currently unknown. However, overexpression of EPOR activates conditions. In addition the survival of SYK-deficient B cells involves cytokine signalling through the JAK-STAT pathway, providing not only BAFF but also CD19-mediated PI3K signalling.92,93 a proliferative advantage in BCP-ALL.78,79 Therefore, developing novel antibody- drug conjugates to target Almost 25% of IGH rearrangements involve the type 1 cytokine CD19 may be useful in the treatment of BCP-ALL as well as B-cell receptor, CRLF2 (cytokine receptor-like factor 2), which forms the lymphoma due to its widespread expression (target presence) and transmembrane receptor for TSLP (thymic stromal-derived lym- potential dependency of the malignant B-lineage cells on its phopoietin) with another type 1 cytokine receptor, IL-7R.80–82 function. Now several antibodies targeting the CD19 antigen are These translocations, as well as juxtaposition of CLRF2 to the available, most of them conjugated to immunotoxins. Blinatumo- promoter of P2RY8 (G-protein coupled Purinergic Receptor P2Y8), mab, the first member of a novel bispecific single-chain antibody, lead to overexpression of CRLF2, which are frequently associated combines a CD3-binding site for T cells and a CD19-binding site with downstream mutations of the JAK gene family.80 In addition for B cells. Experimental models and phase I clinical trials have to the translocations, the general mechanism that activates the shown promising results. In fact, a recent phase 1/II study in type I cytokine receptors such as CLRF2 and IL-7R is shown to be paediatric patients with Relapsed/Refractory BCP-ALL showed the addition of cysteine to their juxtamembranous domains.83 antitumor activity of blinatumomab treatment.94,95 Although Together they activate the JAK-STAT pathway, as well as the these data demonstrate antibody therapy in ALL as a promising PI3K/mTOR pro-proliferative and pro-survival pathways.80,84 approach, its exact role in the treatment of ALL remains to be In a recently identified poor-risk subtype of BCP-ALL, known as determined and may depend on the level of antigen expression BCR-ABL1-like or Ph-like ALL,85,86 chromosomal rearrangements on the leukaemia blasts, stage of the disease and most involving ABL1, ABL2, JAK2 and PDGFRB (platelet-derived growth importantly the residual T-cell function in the patient.96 factor receptor, beta polypeptide) and EPOR were shown to Specific inhibitors of critical players in pre-BCR signalling in facilitate leukaemic transformation by inducing constitutive kinase clinical development include inhibitors of SYK, BTK and PI3K. For activation and signalling through the activation of ABL1 and/or example, the ATP mimetic inhibitor of SYK, fostamatinib (R788), JAK-STAT pathways.78,87 Mutations of JAK2, IL-7R and FLT3 (Fms- which was initially developed for the treatment of inflammatory Related Tyrosine Kinase 3) activate the same pro-proliferative diseases, has been shown to be effective in B-cell malignancies. In pathways.78,88 completed fostamatinib Phase I and II clinical trials, patients This plethora of novel findings indicates that our knowledge showed prolonged survival, however management of toxicity of the genetic complexity of BCP-ALL development remains continues to be a challenge.97–99 Recently, other more specific SYK incomplete. In light of the emerging literature on pre-BCR and inhibitors, such as PRT318 and P505-15, have been tested in BCP-ALL signalling, it is not surprising that the majority of genetic chronic lymphocytic leukaemia, as well as other B-cell malig- modifications occur within pre-BCR-mediated key pathways that nancies although their clinical application remains to be control B-cell survival, proliferation and differentiation. established.99 In addition to these direct mechanisms, SYK hyperactivation, which mimics the acute activation of a self-reactive BCR, has also TARGETING PRE-BCR AND THE ASSOCIATED SIGNALLING been shown to induce cell death in Ph-positive BCP-ALL cells.100 PATHWAY MOLECULES The pharmacological hyperactivation of SYK is achieved by Despite vast improvements in survival of patients with BCP-ALL, inhibiting SHIP1 phosphatase using the small molecule inhibitor, the outcome remains dismal for patients who relapse, due to their 3-α-aminocholestane. SHIP1 phosphatase regulates the SYK resistance to current treatment. For this reason, as well as the inhibitory receptors, PECAM1, CD300A and LAIR1, which are knowledge that current chemotherapy is associated with highly expressed in BCP-ALL. Thus, targeted overexpression of significant acute and long-term toxicity, there is a need for SYK, by blocking its negative regulators, has been shown to be development of targeted therapies. In mature B-cell malignancies sufficient to induce cell death in Ph-positive ALL cells.100 active BCR signalling offers opportunities for therapeutic inter- A number of BTK inhibitors have recently entered clinical trials vention. In contrast, in BCP-ALL the different ways in which the for B-cell malignancies.101,102 Dasatinib (BMS-354825; Spycell), a leukaemic cells hijack or circumvent pre-BCR signalling may offer multi kinase type I inhibitor, initially developed to target BCR-ABL1 novel therapeutic opportunities. fusion kinase, has also been shown to inhibit BTK efficiently. In pre-BCR positive BCP-ALL, pre-BCR and its downstream Clinical trials of dasatinib in the treatment of BCP-ALL are being signalling components, such as CD19, SYK, BTK and PI3K, provide established for inhibition of ABL fusion kinases (see below). More attractive therapeutic interventions. Recently, when pre-BCR selective, covalently binding, ATP mimetic, type II inhibitors, such positive (n = 8) and pre-BCR negative (n = 9) patient-derived ALL as ibrutinib (PCI-32765) and AVL-292, have been shown to samples were treated with a diverse panel of 51 kinase inhibitors, irreversibly inhibit BTK. pre-BCR positive ALL cells were shown to be particularly sensitive A range of inhibitors of PI3K signalling are in clinical trial.103 to inhibitors of SYK (PRT062607), BTK (ibrutinib, dasatinib) and Among them, GS-1101 is a potent inhibitor that specifically blocks PIK3δ.20 In contrast, pre-BCR-negative ALL cells were most the PI3K subtype, PI3Kδ. In addition to these specific isoform vulnerable to inhibition of ABL1, PDGFR, ERK1, ERK2, MET and inhibitors, development of pan PI3K inhibitors, such as VP-BKM120 KIT kinase activity, indicating that different signalling mechanisms from Novartis, are also ongoing. have a central role dependent on the type of oncogenic signalling Genes involved in the RAS-RAF-MEK-ERK signal transduction regulators involved in the development of specific BCP-ALL cascade are also being pursued as drug targets. Constitutive subtype.20,89 activation of this signalling cascade is particularly frequent in

Leukemia (2015) 1623 – 1631 © 2015 Macmillan Publishers Limited pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1629 childhood BCP-ALL. For example, mutations in KRAS (G12D) are REFERENCES common, while, mutations in FLT3, PTPN11, c-CBL, BRAF and 1 Moorman AV. The clinical relevance of chromosomal and genomic abnormalities – NF1 genes are also reported.104 114 Thus RAF-MEK-ERK pathway in B-cell precursor acute lymphoblastic leukaemia. Blood Rev 2012; 26:123–135. molecules provide opportunities for therapeutic intervention. The 2 Pui CH, Mullighan CG, Evans WE, Relling MV. Pediatric acute lymphoblastic 120 potent MEK1/2 inhibitor, selumetinib (AZD6244, ARRY-142886), leukemia: where are we going and how do we get there? Blood 2012; : 1165–1174. showed activity in an orthotopic mouse model engrafted with RAS 3 Roberts KG, Mullighan CG. How new advances in genetic analysis are influencing pathway mutated BCP-ALL cells, with associated reduction in the understanding and treatment of childhood acute leukemia. Curr Opin Pediatr p-ERK and increased levels of the apoptotic biomarkers, Bim and 2011; 23:34–40. cleaved PARP.115 These findings suggest that targeted MEK 4 Harrison CJ, Haas O, Harbott J, Biondi A, Stanulla M, Trka J et al. Detection of inhibition with selumetinib is a possible new therapeutic approach prognostically relevant genetic abnormalities in childhood B-cell precursor acute for BCP-ALL patients with RAS pathway mutations. lymphoblastic leukaemia: recommendations from the Biology and Diagnosis fi Committee of the International Berlin-Frankfurt-Munster study group. Br J This theme of differential BCP-ALL-speci c signalling regulating 151 – fl Haematol 2010; :132 142. BCP-ALL development and therapy is re ected in high-risk, Ph-like 5 Zhang J, Mullighan CG, Harvey RC, Wu G, Chen X, Edmonson M et al. ALL. This subtype shares same gene expression profile and poor Key pathways are frequently mutated in high-risk childhood acute lymphoblastic outcome of BCR-ABL1 positive ALL, but lacks the BCR-ABL1 fusion leukemia: a report from the Children's Oncology Group. Blood 2011; 118: protein.85,86 It harbours a diverse range of alterations that activate 3080–3087. cytokine receptors (CRLF2, EPOR and JAK2)78 or tyrosine kinases 6 Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD et al. such as ABL1, ABL2, TSLP, CSF1R, NTRK3, PDGFRB, PTK2B or TYK2 Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 2007; 446:758–764. and sequence mutations involving FLT3, IL-7R or SH2B3, among 7 Moorman AV, Enshaei A, Schwab C, Wade R, Chilton L, Elliott A et al. A novel 87 others. The main mechanism used by these genes that enable integrated cytogenetic and genomic classification refines risk stratification in the leukaemic transformation is subversion of Ras and JAK/STAT5 pediatric acute lymphoblastic leukemia (ALL). Blood 2014; 124: 1434–1444. pathways.78,116 Preclinical studies and limited clinical experience 8 le Viseur C, Hotfilder M, Bomken S, Wilson K, Rottgers S, Schrauder A et al. In indicate that these leukaemias respond to tyrosine kinase childhood acute lymphoblastic leukemia, blasts at different stages of immuno- 14 – inhibitors such as dasatinib and ruxolitinib.87,117 From these phenotypic maturation have stem cell properties. Cancer Cell 2008; :47 58. fi 9 Rehe K, Wilson K, Bomken S, Williamson D, Irving J, den Boer ML et al. Acute B examples, evidence is accumulating that BCP-ALL-speci c signal- lymphoblastic leukaemia-propagating cells are present at high frequency in ling molecules provide promising new drug targets. diverse lymphoblast populations. EMBO Mol Med 2013; 5:38–51. 10 Dick JE. Stem cell concepts renew cancer research. Blood 2008; 112: 4793–4807. 11 Herzog S, Reth M, Jumaa H. Regulation of B-cell proliferation and differentiation SUMMARY by pre-B-cell receptor signalling. Nat Rev Immunol 2009; 9: 195–205. The pre-BCR checkpoint and the signals emanating from it are 12 Matthias P, Rolink AG. Transcriptional networks in developing and mature B cells. 5 – emerging as major regulatory switches that regulate clonal rescue Nat Rev Immunol 2005; : 497 508. 13 Rickert RC. New insights into pre-BCR and BCR signalling with relevance to B cell and expansion in BCP-ALL. Therefore it is not surprising that the malignancies. Nat Rev Immunol 2013; 13: 578–591. most prevalent genetic aberrations; fusion oncogenes, deletions 14 Melchers F. The pre-B-cell receptor: selector of fitting immunoglobulin heavy and point mutations, are found in key molecules that regulate cell chains for the B-cell repertoire. Nat Rev Immunol 2005; 5:578–584. survival, proliferation and differentiation, modulating the pre-BCR 15 Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient mouse by targeted checkpoint. disruption of the membrane exon of the immunoglobulin mu chain gene. It also needs to be understood how communication between Nature 1991; 350: 423–426. the microenvironment and pre-BCR are manipulated in BCP-ALL. 16 Hendriks RW, Middendorp S. The pre-BCR checkpoint as a cell-autonomous proliferation switch. Trends Immunol 2004; 25:249–256. For example, are membrane receptors, such as CRLF2, activated to 17 Clark MR, Mandal M, Ochiai K, Singh H. Orchestrating B cell lymphopoiesis enable the pre-BCR checkpoint to be bypassed? How important through interplay of IL-7 receptor and pre-B cell receptor signalling. Nat Rev are the niche survival signals and can they be exploited Immunol 2014; 14:69–80. therapeutically? As there is significant plasticity in cell growth 18 Kuppers R. Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer 2005; and survival pathways, the key challenge for BCP-ALL is to develop 5:251–262. rationally designed combination strategies that efficiently and 19 Young RM, Staudt LM. Targeting pathological B cell receptor signalling in lymphoid malignancies. Nat Rev Drug Discov 2013; 12:229–243. selectively kill the leukaemic cells and prevent the emergence of 20 Geng H, Hurtz C, Lenz KB, Chen Z, Baumjohann D, Thompson S et al. Self- resistance. Therefore, elucidation of those leukaemic signalling enforcing feedback activation between BCL6 and pre-B cell receptor signaling networks that promote pre B-cell transformation and leukaemic defines a distinct subtype of acute lymphoblastic leukemia. Cancer Cell 2015; 27: maintenance at the pre-BCR checkpoint will not only enhance our 409–425. understanding of BCP-ALL evolution but also identify new targets 21 Schmitz R, Young RM, Ceribelli M, Jhavar S, Xiao W, Zhang M et al. Burkitt that offer realistic therapeutic development opportunities. Most lymphoma pathogenesis and therapeutic targets from structural and functional genomics. Nature 2012; 490:116–120. importantly, understanding how the different subtypes of ALL 22 Burger JA, Okkenhaug K. Haematological cancer: idelalisib-targeting PI3Kdelta in hijack and/or circumvent the pre-BCR checkpoint will guide future patients with B-cell malignancies. Nat Rev Clin Oncol 2014; 11: 184–186. treatment with novel targeted therapies interfering with pre-BCR 23 Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB et al. Chronic active signalling. B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010; 463: 88–92. 24 Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA et al. Targeting BTK CONFLICT OF INTEREST with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 2013; 369: 32–42. The authors declare no conflict of interests. 25 Kanzler H, Kuppers R, Hansmann ML, Rajewsky K. Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone ACKNOWLEDGEMENTS derived from (crippled) germinal center B cells. J Exp Med 1996; 184: 1495–1505. 26 Leithauser F, Bauerle M, Huynh MQ, Moller P. Isotype-switched immunoglobulin We are grateful for financial support from: Marie Curie International Incoming genes with a high load of somatic hypermutation and lack of ongoing muta- Fellowship (JE), European Research Council, Leukaemia and Lymphoma Research, tional activity are prevalent in mediastinal B-cell lymphoma. Blood 2001; 98: Cancer Research UK, Kay Kendall Leukaemia Fund and Medical Research Council 2762–2770. career development award MR/J008060/1 (DPC), the programme grant from CR‐UK 27 Hobeika E, Nielsen PJ, Medgyesi D. Signaling mechanisms regulating B-lym- (grant number C27943/A12788). phocyte activation and tolerance. J Mol Med 2015; 93:143–158.

© 2015 Macmillan Publishers Limited Leukemia (2015) 1623 – 1631 pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1630 28 Klein F, Feldhahn N, Muschen M. Interference of BCR-ABL1 kinase activity with 52 Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K. A critical role of antigen receptor signaling in B cell precursor leukemia cells. Cell Cycle 2004; 3: lambda 5 protein in B cell development. Cell 1992; 69:823–831. 858–860. 53 Bradl H, Jack HM. Surrogate light chain-mediated interaction of a soluble pre-B 29 Klein F, Feldhahn N, Harder L, Wang H, Wartenberg M, Hofmann WK et al. The cell receptor with adherent cell lines. J Immunol 2001; 167: 6403–6411. BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in 54 Gauthier L, Rossi B, Roux F, Termine E, Schiff C. Galectin-1 is a stromal cell ligand pre-B acute lymphoblastic leukemia cells. J Exp Med 2004; 199: 673–685. of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B 30 Trageser D, Iacobucci I, Nahar R, Duy C, von Levetzow G, Klemm L et al. Pre-B cell and stromal cells and in pre-BCR triggering. Proc Natl Acad Sci USA 2002; 99: receptor-mediated cell cycle arrest in Philadelphia chromosome-positive acute 13014–13019. lymphoblastic leukemia requires IKAROS function. J Exp Med 2009; 206: 55 Monroe JG. ITAM-mediated tonic signalling through pre-BCR and BCR com- 1739–1753. plexes. Nat Rev Immunol 2006; 6: 283–294. 31 Mangum DS, Downie J, Mason CC, Jahromi MS, Joshi D, Rodic V et al. VPREB1 56 Kohler F, Hug E, Eschbach C, Meixlsperger S, Hobeika E, Kofer J et al. Autoreactive deletions occur independent of lambda light chain rearrangement in childhood B cell receptors mimic autonomous pre-B cell receptor signaling and induce acute lymphoblastic leukemia. Leukemia 2014; 28:216–220. proliferation of early B cells. Immunity 2008; 29:912–921. 32 Swaminathan S, Huang C, Geng H, Chen Z, Harvey R, Kang H et al. BACH2 57 Ubelhart R, Bach MP, Eschbach C, Wossning T, Reth M, Jumaa H. N-linked mediates negative selection and p53-dependent tumor suppression at the pre-B glycosylation selectively regulates autonomous precursor BCR function. Nat cell receptor checkpoint. Nat Med 2013; 19: 1014–1022. Immunol 2010; 11: 759–765. 33 Oyake T, Itoh K, Motohashi H, Hayashi N, Hoshino H, Nishizawa M et al. Bach 58 Su YW, Jumaa H. LAT links the pre-BCR to calcium signaling. Immunity 2003; 19: proteins belong to a novel family of BTB-basic leucine zipper transcription fac- 295–305. tors that interact with MafK and regulate transcription through the NF-E2 site. 59 Yasuda T, Sanjo H, Pages G, Kawano Y, Karasuyama H, Pouyssegur J et al. Mol Cell Biol 1996; 16: 6083–6095. Erk kinases link pre-B cell receptor signaling to transcriptional events required 34 Kawamata N, Pennella MA, Woo JL, Berk AJ, Koeffler HP. Dominant-negative for early B cell expansion. Immunity 2008; 28: 499–508. mechanism of leukemogenic PAX5 fusions. Oncogene 2012; 31: 966–977. 60 Iritani BM, Forbush KA, Farrar MA, Perlmutter RM. Control of B cell development 35 An Q, Wright SL, Konn ZJ, Matheson E, Minto L, Moorman AV et al. Variable by Ras-mediated activation of Raf. EMBO J 1997; 16: 7019–7031. breakpoints target PAX5 in patients with dicentric chromosomes: a model for 61 Wossning T, Herzog S, Kohler F, Meixlsperger S, Kulathu Y, Mittler G et al. the basis of unbalanced translocations in cancer. Proc Natl Acad Sci USA 2008; Deregulated Syk inhibits differentiation and induces growth factor-independent 105: 17050–17054. proliferation of pre-B cells. J Exp Med 2006; 203: 2829–2840. 36 Fazio G, Cazzaniga V, Palmi C, Galbiati M, Giordan M, te Kronnie G et al. 62 Uckun FM, Qazi S. SYK as a new therapeutic target in B-cell precursor acute PAX5/ETV6 alters the gene expression profile of precursor B cells with opposite lymphoblastic leukemia. J Cancer Ther 2014; 5:124–131. dominant effect on endogenous PAX5. Leukemia 2013; 27: 992–995. 63 Grawunder U, Leu TM, Schatz DG, Werner A, Rolink AG, Melchers F et al. Down- 37 Ta VB, de Bruijn MJ, ter Brugge PJ, van Hamburg JP, Diepstraten HJ, van Loo PF regulation of RAG1 and RAG2 gene expression in preB cells after functional et al. Malignant transformation of Slp65-deficient pre-B cells involves disruption immunoglobulin heavy chain rearrangement. Immunity 1995; 3:601–608. of the Arf-Mdm2-p53 tumor suppressor pathway. Blood 2010; 115: 1385–1393. 64 Ochiai K, Maienschein-Cline M, Mandal M, Triggs JR, Bertolino E, Sciammas R 38 Jumaa H, Bossaller L, Portugal K, Storch B, Lotz M, Flemming A et al. Deficiency of et al. A self-reinforcing regulatory network triggered by limiting IL-7 activates the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia. Nature 2003; pre-BCR signaling and differentiation. Nat Immunol 2012; 13:300–307. 423: 452–456. 65 Amin RH, Schlissel MS. Foxo1 directly regulates the transcription of 39 Flemming A, Brummer T, Reth M, Jumaa H. The adaptor protein SLP-65 acts as a recombination-activating genes during B cell development. Nat Immunol 2008; tumor suppressor that limits pre-B cell expansion. Nat Immunol 2003; 4:38–43. 9:613–622. 40 Meixlsperger S, Kohler F, Wossning T, Reppel M, Muschen M, Jumaa H. Con- 66 Feldhahn N, Klein F, Mooster JL, Hadweh P, Sprangers M, Wartenberg M et al. ventional light chains inhibit the autonomous signaling capacity of the B cell Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic receptor. Immunity 2007; 26:323–333. leukemia cells. J Exp Med 2005; 201: 1837–1852. 41 Kersseboom R, Middendorp S, Dingjan GM, Dahlenborg K, Reth M, Jumaa H et al. 67 Duy C, Yu JJ, Nahar R, Swaminathan S, Kweon SM, Polo JM et al. BCL6 is critical Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a for the development of a diverse primary B cell repertoire. J Exp Med 2010; 207: tumor suppressor in Pre-B cells. J Exp Med 2003; 198:91–98. 1209–1221. 42 Dengler HS, Baracho GV, Omori SA, Bruckner S, Arden KC, Castrillon DH et al. 68 Duy C, Hurtz C, Shojaee S, Cerchietti L, Geng H, Swaminathan S et al. BCL6 Distinct functions for the transcription factor Foxo1 at various stages of B cell enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase differentiation. Nat Immunol 2008; 9:1388–1398. inhibition. Nature 2011; 473:384–388. 43 Nakayama J, Yamamoto M, Hayashi K, Satoh H, Bundo K, Kubo M et al. BLNK 69 Geng H, Brennan S, Milne TA, Chen WY, Li Y, Hurtz C et al. Integrative epige- suppresses pre-B-cell leukemogenesis through inhibition of JAK3. Blood 2009; nomic analysis identifies biomarkers and therapeutic targets in adult B-acute 113: 1483–1492. lymphoblastic leukemia. Cancer Discov 2012; 2:1004–1023. 44 Nahar R, Ramezani-Rad P, Mossner M, Duy C, Cerchietti L, Geng H et al. Pre-B cell 70 Bicocca VT, Chang BH, Masouleh BK, Muschen M, Loriaux MM, Druker BJ et al. receptor-mediated activation of BCL6 induces pre-B cell quiescence through Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) transcriptional repression of MYC. Blood 2011; 118:4174–4178. acute lymphoblastic leukemia. Cancer Cell 2012; 22: 656–667. 45 Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. BCR-ABL1 71 van der Veer A, van der Velden VH, Willemse ME, Hoogeveen PG, Petricoin EF, lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008; Beverloo HB et al. Interference with pre-B-cell receptor signaling offers a ther- 453: 110–114. apeutic option for TCF3-rearranged childhood acute lymphoblastic leukemia. 46 Imai C, Ross ME, Reid G, Coustan-Smith E, Schultz KR, Pui CH et al. Expression of Blood Cancer J 2014; 4:e181. the adaptor protein BLNK/SLP-65 in childhood acute lymphoblastic leukemia. 72 Broome HE, Rassenti LZ, Wang HY, Meyer LM, Kipps TJ. ROR1 is expressed on Leukemia 2004; 18:922–925. hematogones (non-neoplastic human B-lymphocyte precursors) and a minority 47 Goodman PA, Wood CM, Vassilev AO, Mao C, Uckun FM. Defective expression of of precursor-B acute lymphoblastic leukemia. Leuk Res 2011; 35: 1390–1394. Bruton's tyrosine kinase in acute lymphoblastic leukemia. Leuk Lymphoma 2003; 73 Fine BM, Stanulla M, Schrappe M, Ho M, Viehmann S, Harbott J et al. Gene 44: 1011–1018. expression patterns associated with recurrent chromosomal translocations in 48 Hoshino A, Okuno Y, Migita M, Ban H, Yang X, Kiyokawa N et al. X-linked acute lymphoblastic leukemia. Blood 2004; 103:1043–1049. agammaglobulinemia associated with B-precursor acute lymphoblastic leuke- 74 Inthal A, Krapf G, Beck D, Joas R, Kauer MO, Orel L et al. Role of the erythropoietin mia. J Clin Immunol 2015; 35:108–111. receptor in ETV6/RUNX1-positive acute lymphoblastic leukemia. Clin Cancer Res 49 Conley ME. Are patients with x-linked agammaglobulinemia at increased 2008; 14:7196–7204. risk of developing acute lymphoblastic leukemia? J Clin Immunol 2015; 35: 75 van Delft FW, Bellotti T, Luo Z, Jones LK, Patel N, Yiannikouris O et al. Prospective 98–99. gene expression analysis accurately subtypes acute leukaemia in children and 50 Sulong S, Moorman AV, Irving JA, Strefford JC, Konn ZJ, Case MC et al. A com- establishes a commonality between hyperdiploidy and t(12;21) in acute prehensive analysis of the CDKN2A gene in childhood acute lymphoblastic lymphoblastic leukaemia. Br J Haematol 2005; 130:26–35. leukemia reveals genomic deletion, copy number neutral loss of heterozygosity, 76 Torrano V, Procter J, Cardus P, Greaves M, Ford AM. ETV6-RUNX1 promotes and association with specific cytogenetic subgroups. Blood 2009; 113:100–107. survival of early B lineage progenitor cells via a dysregulated erythropoietin 51 Zachariadis V, Schoumans J, Barbany G, Heyman M, Forestier E, Johansson B receptor. Blood 2011; 118: 4910–4918. et al. Homozygous deletions of CDKN2A are present in all dic(9;20)(p13.2;q11.2)- 77 Russell LJ, Akasaka T, Majid A, Sugimoto KJ, Loraine Karran E, Nagel I et al. t(6;14) positive B-cell precursor acute lymphoblastic leukaemias and may be important (p22;q32): a new recurrent IGH@ translocation involving ID4 in B-cell precursor for leukaemic transformation. Br J Haematol 2012; 159:488–491. acute lymphoblastic leukemia (BCP-ALL). Blood 2008; 111:387–391.

Leukemia (2015) 1623 – 1631 © 2015 Macmillan Publishers Limited pre-B-cell receptor checkpoint in leukaemia J Eswaran et al 1631 78 Roberts KG, Morin RD, Zhang J, Hirst M, Zhao Y, Su X et al. Genetic alterations 98 Chen L, Monti S, Juszczynski P, Daley J, Chen W, Witzig TE et al. SYK-dependent activating kinase and cytokine receptor signaling in high-risk acute lympho- tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell blastic leukemia. Cancer Cell 2012; 22: 153–166. lymphoma. Blood 2008; 111: 2230–2237. 79 Russell LJ, De Castro DG, Griffiths M, Telford N, Bernard O, Panzer-Grumayer R 99 Hoellenriegel J, Coffey GP, Sinha U, Pandey A, Sivina M, Ferrajoli A et al. Selective, et al. A novel translocation, t(14;19)(q32;p13), involving IGH@ and the cytokine novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leu- receptor for erythropoietin. Leukemia 2009; 23:614–617. kemia B-cell activation and migration. Leukemia 2012; 26:1576–1583. 80 Russell LJ, Capasso M, Vater I, Akasaka T, Bernard OA, Calasanz MJ et al. 100 Chen Z, Shojaee S, Buchner M, Geng H, Lee JW, Klemm L et al. Signalling Deregulated expression of cytokine receptor gene, CRLF2, is involved in thresholds and negative B-cell selection in acute lymphoblastic leukaemia. lymphoid transformation in B-cell precursor acute lymphoblastic leukemia. Blood Nature 2015; 521:357–361. 2009; 114: 2688–2698. 101 Burger JA, Buggy JJ. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765). Leuk 81 Russell LJ, Enshaei A, Jones L, Erhorn A, Masic D, Bentley H et al. IGH@ Trans- Lymphoma 2013; 54: 2385–2391. locations Are Prevalent in Teenagers and Young Adults With Acute Lympho- 102 Hendriks RW. Drug discovery: new Btk inhibitor holds promise. Nat Chem Biol blastic Leukemia and Are Associated With a Poor Outcome. J Clin Oncol 2014; 32: 2011; 7:4–5. 1453–1462. 103 Fruman DA, Rommel C. PI3Kdelta inhibitors in cancer: rationale and serendipity 82 Moorman AV, Schwab C, Ensor HM, Russell LJ, Morrison H, Jones L et al. IGH@ merge in the clinic. Cancer Discov 2011; 1: 562–572. translocations, CRLF2 deregulation, and microdeletions in adolescents and 104 Wiemels JL, Zhang Y, Chang J, Zheng S, Metayer C, Zhang L et al. RAS mutation is adults with acute lymphoblastic leukemia. J Clin Oncol 2012; 30: 3100–3108. associated with hyperdiploidy and parental characteristics in pediatric acute 83 Shochat C, Tal N, Bandapalli OR, Palmi C, Ganmore I, te Kronnie G et al. Gain-of- lymphoblastic leukemia. Leukemia 2005; 19:415–419. function mutations in interleukin-7 receptor-alpha (IL7R) in childhood acute 105 Perentesis JP, Bhatia S, Boyle E, Shao Y, Shu XO, Steinbuch M et al. RAS oncogene lymphoblastic leukemias. J Exp Med 2011; 208:901–908. mutations and outcome of therapy for childhood acute lymphoblastic leukemia. 84 Tasian SK, Doral MY, Borowitz MJ, Wood BL, Chen IM, Harvey RC et al. Aberrant Leukemia 2004; 18:685–692. STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2-rearranged 106 Tartaglia M, Martinelli S, Cazzaniga G, Cordeddu V, Iavarone I, Spinelli M et al. B-precursor acute lymphoblastic leukemia. Blood 2012; 120: 833–842. Genetic evidence for lineage-related and differentiation stage-related con- 85 Den Boer ML, van Slegtenhorst M, De Menezes RX, Cheok MH, Buijs-Gladdines tribution of somatic PTPN11 mutations to leukemogenesis in childhood acute JG, Peters ST et al. A subtype of childhood acute lymphoblastic leukaemia with leukemia. Blood 2004; 104:307–313. poor treatment outcome: a genome-wide classification study. Lancet Oncol 2009; 107 Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA et al. FLT3 10: 125–134. mutations in childhood acute lymphoblastic leukemia. Blood 2004; 103: 86 Mullighan CG, Su X, Zhang J, Radtke I, Phillips LA, Miller CB et al. Deletion of 3544–3546. IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med 2009; 360: 108 Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R et al. FLT3 mutations in 470–480. the activation loop of tyrosine kinase domain are frequently found in infant ALL 87 Roberts KG, Li Y, Payne-Turner D, Harvey RC, Yang YL, Pei D et al. Targetable with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood 2004; kinase-activating lesions in Ph-like acute lymphoblastic leukemia. N Engl J Med 103: 1085–1088. 2014; 371: 1005–1015. 109 Gustafsson B, Angelini S, Sander B, Christensson B, Hemminki K, Kumar R. 88 Loh ML, Zhang J, Harvey RC, Roberts K, Payne-Turner D, Kang H et al. Tyrosine Mutations in the BRAF and N-ras genes in childhood acute lymphoblastic leu- kinome sequencing of pediatric acute lymphoblastic leukemia: a report from the kaemia. Leukemia 2005; 19:310–312. Children's Oncology Group TARGET Project. Blood 2013; 121:485–488. 110 Case M, Matheson E, Minto L, Hassan R, Harrison CJ, Bown N et al. Mutation of 89 Trimarchi T, Aifantis I. The pre-BCR to the rescue: therapeutic targeting of pre-B genes affecting the RAS pathway is common in childhood acute lymphoblastic cell ALL. Cancer Cell 2015; 27:321–323. leukemia. Cancer Res 2008; 68: 6803–6809. 90 Weiland J, Elder A, Forster V, Heidenreich O, Koschmieder S, Vormoor J. CD19: 111 Nicholson L, Knight T, Matheson E, Minto L, Case M, Sanichar M et al. Casitas B a multifunctional immunological target molecule and its implications for Bline- lymphoma mutations in childhood acute lymphoblastic leukemia. Genes Chro- age acute lymphoblastic leukemia. Pediatr Blood Cancer 2015. mosomes Cancer 2012; 51:250–256. 91 Geng H, Chen Z, Park E, Klemm L, Bailey CC, Muschen M. Ifitm3 (CD225) 112 Molteni CG, Te Kronnie G, Bicciato S, Villa T, Tartaglia M, Basso G et al. mediates CD19-dependent survival and proliferation during normal B cell PTPN11 mutations in childhood acute lymphoblastic leukemia occur as a development and In Ph+ ALL. Blood J 2013; vol. 122: 2505–2505. secondary event associated with high hyperdiploidy. Leukemia 2010; 24: 92 Hobeika E, Levit-Zerdoun E, Anastasopoulou V, Pohlmeyer R, Altmeier S, Alsadeq 232–235. A et al. CD19 and BAFF-R can signal to promote B-cell survival in the absence of 113 Balgobind BV, Van Vlierberghe P, van den Ouweland AM, Beverloo HB, Terlouw- Syk. Embo J 2015; 34: 925–939. Kromosoeto JN, van Wering ER et al. Leukemia-associated NF1 inactivation in 93 Konigsberger S, Kiefer F. The BAFFling function of Syk in B-cell homeostasis. patients with pediatric T-ALL and AML lacking evidence for neurofibromatosis. EMBO J 2015; 34: 838–840. Blood 2008; 111: 4322–4328. 94 Stackelberg Av. A phase 1/2 study of blinatumomab in pediatric patients with 114 Yamamoto T, Isomura M, Xu Y, Liang J, Yagasaki H, Kamachi Y et al. PTPN11, RAS relapsed/refractory B-cell precursor acute lymphoblastic leukemia. Blood 2013; and FLT3 mutations in childhood acute lymphoblastic leukemia. Leuk Res 2006; 122: 70. 30: 1085–1089. 95 Schlegel P, Lang P, Zugmaier G, Ebinger M, Kreyenberg H, Witte KE et al. 115 Irving J, Matheson E, Minto L, Blair H, Case M, Halsey C et al. Ras pathway Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic mutations are highly prevalent in relapsed childhood acute lymphoblastic leu- leukemia shows durable remission by therapy with the T-cell engaging bispecific kaemia, may act as relapse-drivers and confer sensitivity to MEK inhibition. Blood antibody blinatumomab. Haematologica 2014; 99: 1212–1219. 2014; 124:3420–3430. 96 Hoelzer D. Novel antibody-based therapies for acute lymphoblastic leukemia. 116 Harrison CJ. Genomic analysis drives tailored therapy in poor risk childhood Hematology Am Soc Hematol Educ Program 2011; 2011:243–249. leukemia. Cancer Cell 2012; 22:139–140. 97 Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, Lacasce A et al. 117 Roberts KG, Pei D, Campana D, Payne-Turner D, Li Y, Cheng C et al. Outcomes of Inhibition of Syk with fostamatinib disodium has significant clinical activity in children with BCR-ABL1-like acute lymphoblastic leukemia treated with risk- non-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood 2010; 115: directed therapy based on the levels of minimal residual disease. J Clin Oncol 2578–2585. 2014; 32: 3012–3020.