Tracing the Pre-B to Immature B Cell Transition in Human Leukemia Cells Reveals a Coordinated Sequence of Primary and Secondary IGK Gene Rea
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免疫学杂志 2005年第1期
Abstract
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, C and C germline transcription, and rearrangement of Ig L chain (IGK) and Ig L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed L, but almost no L chains. This could be explained by STI571-induced rearrangement of the -deleting element (KDE), which can delete productively rearranged V-J joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of V-J joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of C and C germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located J- to downstream J-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.
Introduction
B cell precursors within the bone marrow undergo a sequence of Ig gene rearrangements defining distinct stages of early B cell differentiation (1). During their early development, B cell precursors have to pass checkpoints at which only cells carrying functional Ig gene rearrangements are selected for further development along the B cell lineage (2). For instance, the presence of a productive Ig μ H chain (IGH)3 gene rearrangement is a prerequisite for the expression of the Ig μ H chain as a component of the pre-B cell receptor on the surface of pre-B cells (PBC). As soon as a B cell precursor successfully rearranges VH, DH, and JH gene segments on one allele, the recombination machinery is halted. This prevents further rearrangement of the second allele, which constitutes allelic exclusion at the IGH locus (3). Termination of the rearrangement process at the IGH locus is followed by the initiation of Ig L chain gene recombination at the Ig L chain (IGK) and Ig L chain (IGL) loci, which defines the pre-B to immature B cell transition. Human B cells express more frequently L than L chains, at a / ratio of 1.4 (4). To explain the relative overrepresentation of IGK gene rearrangements, which is even more striking in mice, two models have been proposed: the "stochastic model" (5, 6) postulates that IGK and IGL loci are independently accessible to recombinase activity and that cis-acting regulatory elements (e.g., the 3' enhancer; Refs. 7 and 8) determine the ratio of L and L chain-expressing cells. This model was supported by the finding that -expressing B cells could arise from mice carrying inactivated IGK loci on both alleles (9, 10). On the contrary, the "ordered model" proposes that IGK genes, as a rule, rearrange before IGL gene recombination (11, 12). Consistent with an ordered model of IGK and IGL gene rearrangement, recent work showed that single human -expressing B cells (13) or B cell precursor leukemia clones (14) harbored inactivated V-J gene rearrangements almost in all instances in addition to productive V-J joints but not vice versa: only few -expressing B cells or leukemic B cell clones also carried inactive V-J joints in addition to productive V-J gene rearrangements. These findings argue for sequential rearrangement of IGK and IGL genes. However, these studies do not formally demonstrate that rearrangement of IGK genes in -expressing B cells do necessarily precede successful V-J gene recombination. In -expressing B cells, for instance, IGL genes may well be rearranged first, followed by needless V-J gene rearrangements, which then would subsequently be inactivated by rearrangement of the -deleting element (KDE). The KDE may rearrange to recombination signal sequences (RSS) flanking germline V gene segments or an RSS within the intron between the J cluster and the C gene, which leads to inactivation of a pre-existing V-J joint in either case (15). Also the existence of B cells expressing L together with L chains (16) suggests that recombination events in the IGK and IGL loci are not mutually exclusive in all instances. Instead of L or L chains, human PBC express surrogate L chains composed of VpreB and 5.
In the vast majority of acute lymphoblastic leukemia (ALL), the malignant clone represents the outgrowth of a transformed PBC. In many cases, pre-B ALL clones carry specific oncogenic gene rearrangements defining both biological and clinical subentities (17). Among these translocation events, the t(9;22)(q34;q11) results in a fusion of the BCR and ABL1 genes, which codes for a constitutive active variant of the ABL1 tyrosine kinase (18) and represents the most frequent recurrent aberration leading to ALL in adults (19). ABL1 represents the human homologue of the transforming gene of the Abelson murine leukemia virus, v-abl. Although v-abl has been used for many years to transform murine PBC, recent work showed that v-abl also confers a differentiation block at the PBC stage of development and, hence, prevents rearrangement of IGK or IGL L chain genes (20, 21). Recently, a specifically designed inhibitor of BCR-ABL1, termed STI571, has become available and is now widely used as an anti-leukemia drug for BCR-ABL1+ leukemias (22). Therefore, we investigated whether and in which way inhibition of BCR-ABL1 kinase activity by STI571 might induce differentiation including IGK and IGL gene rearrangement in pre-B ALL cells.
Materials and Methods
Patient samples, primary cells, and cell lines
BCR-ABL1+ pre-B ALL cells from five patients were analyzed. From three of these patients, leukemia cells were available before and during treatment with STI571 (cases III, IV, and V, Refs. 23 and 24). Samples of two other cases (cases I and II, Ref. 24) were used for serial analysis of gene expression (SAGE) analysis. Normal human CD10+VpreB+ PBC, CD34+CD38lowCD133+ hemopoietic progenitor cells (HSC),CD15+CD34– myeloid progenitor cells, CD7+CD10+ T lymphoid progenitor cells (TLP) and mature B cell subsets including CD19+CD27– naive B cells (NBC), CD20+CD77+ germinal center B cells (GCB), CD19+CD27+ memory B cells (MBC), and CD19lowCD138+ plasma cells (PC) were isolated by MACS and FACS from bone marrow, umbilical cord blood, peripheral blood, or tonsils from healthy donors as previously described (25, 26, 27, 28). For cell culture experiments, the BCR-ABL1+ pre-B ALL cell lines BV173, NALM1, and SUP-B15 (Deutsche Sammlung von Mikroorganismen und Zellkulturen) were used. Cell lines were treated with 10 μmol/L STI571 (Novartis) for the times indicated.
SAGE analysis
To identify differentially expressed genes between BCR-ABL1+ pre-B ALL cells and normal PBC that play a role in Ig L chain gene regulation, we analyzed mRNA expression profiles generated by the SAGE method. A total of 592,000 SAGE tags were collected for 10 SAGE profiles. A total of 106,000 tags were analyzed from the CD34+ HSC library, 99,000 for CD15+ common myeloid progenitor cells (CMP), 110,000 for CD10+CD19+ PBC, 96,500 for CD7+CD10+ TLP, and each 30,000 tags for two cases of bone marrow-derived pre-B ALL carrying a BCR-ABL1 gene rearrangement (cases I and II), CD19+CD27– NBC, CD20+ CD77+ GCB, CD19+CD27+ MBC, and CD19+CD138+ PC (see Fig. 1A). All SAGE libraries were normalized to 100,000 tags. SAGE data were graphically visualized using the Cluster and Treeview software (http://rana.lbl.gov/) and sorted according to the ratio between SAGE-tag counts in PBC and in BCR-ABL1+ ALL cases (26, 27, 28). In these SAGE profiles, 30 genes were identified which have been implicated in the regulation of Ig L chain expression. These genes were searched in the literature, as well as in UniGene () and OMIM () databases.
FIGURE 1. mRNA levels of Ig L chain-associated genes in BCR-ABL1+ pre-B ALL cells. A, Focusing on 30 genes implicated in the regulation of Ig L chain expression, genome-wide SAGE profiles of two BCR-ABL1+ pre-B ALL cases (ALL1 and ALL2) were compared with SAGE profiles of normal CD34+CD38low HSC, CD15+ CMP, CD7+CD10+ TLP, CD10+CD19+ PBC, CD19+CD27– NBC, CD20+CD77+ GCB, CD19+CD27+ MBC, and CD19lowCD138+ PC. SAGE-tag counts are visualized by colors, red indicating high and green depicting low levels or no expression, respectively. SAGE data were sorted according to the ratio of SAGE-tag counts in normal B cell subsets to leukemia samples. B, From three patients with BCR-ABL1+ pre-B ALL, leukemia samples were available before and during treatment with the BCR-ABL1 kinase inhibitor STI571. In these matched leukemia sample pairs, mRNA levels of IRF4 and SPIB were compared by semiquantitative RT-PCR. cDNA amounts were normalized by OD measurements and amplification of a specific fragment of the housekeeping gene GAPDH.
RT-PCR analysis of IRF4, SPIB, and RAG1 and RAG2 expression and germline C and C transcripts
mRNA levels of the Ig L chain-associated transcription factors IRF4 and SPIB were measured in BCR-ABL1+ leukemia samples from three patients (cases III, IV, and V, Ref. 24) before and during treatment with STI571 (see Fig. 1). From patient samples, total RNA was isolated and transcribed into cDNA as previously described (26, 27, 28). Amounts of cDNA were normalized by OD measurement and semiquantitative RT-PCR using 5'-TTAGCACCCCTGGCCAAG-3' and 5'-CTTACTCCTTGGAGGCCATG-3' for amplification of a cDNA fragment of GAPDH. For semiquantitative RT-PCR analysis of IRF4 the oligonucleotides 5'-CAAGAGCAATGACTTTGAGG-3' and 5'-TGGGACATTGGTACGGGAT-3' were used and for SPIB 5'-AAGACTTACCGTTGGACAGC-3' and 5'-CTTGGAGGAGAACTGGAAGA-3'.
To determine L chain locus germline transcription by RT-PCR, RNA was isolated from BV173, NALM1, and SUP-B15 cell lines with or without STI571 incubation, reverse-transcribed into cDNA, and normalized as described above. For amplification of C germline transcripts, 5'-TTCAACAGGGGAGAGTGTTAGAG-3' and 5'-ATGCGCCTTAGGATGACTACATA-3' primers were used, and 5'-TGTCTGATCAGTGACTTCTACCC-3' and 5'-CTGTAGCTTCTGTGGGACTTC-3' primers were used for C germline transcripts. RT-PCR for RAG1 and RAG2 was conducted using the oligonucleotides 5'-ATAGAAGAAAGCAACACAAAAGC-3' and 5'-ATACTGAGTTCAATCCCTGAAGA-3' for RAG1 and 5'-ATAGCAAGAGCTCTACACACTCC-3' and 5'-AAAAATCAGATCAGAAATCCTCA-3' for RAG2 (see Fig. 2A).
FIGURE 2. Inhibition of BCR-ABL1 by STI571 results in C and C germline transcription and rapid up-regulation of RAG1 and RAG2. Three BCR-ABL1+ pre-B ALL cell lines were incubated in the presence or absence of 10 μmol/L STI571 for the times indicated and analyzed for the expression of C and C germline transcription, RAG1, RAG2, and GAPDH (control) by RT-PCR (A). Nuclear RAG1 expression in individual leukemia cells was measured by staining for CD19 (green) and intracellular RAG1 (red; B).
Analysis of nuclear RAG1 expression
BCR-ABL1+ pre-B ALL cells (BV173 and SUP-B15) were cultured in the presence or absence of STI571 for 1, 2, or 4 h and subsequently stained with a Cy5-labeled Ab against CD19. After treatment with methanol and 4% paraformaldehyde, cells were stained with a mouse anti-human RAG1 IgG1 (BD Biosciences) together with a Cy2-labeled goat anti-mouse IgG Ab. Expression of CD19 and RAG1 was visualized by immunofluorescence (see Fig. 2B).
Flow cytometry
Surface expression of IGK or IGL on BV173, NALM1, and SUP-B15 pre-B ALL cells in the presence or absence of STI571 was monitored using Abs against IGK or IGL (BD Biosciences) after the incubation times indicated (see Fig. 3). Apoptotic or dead cells were identified by staining with FITC-labeled annexin V and propidium iodide (BD Biosciences) and excluded from analysis. Normal B cells were enriched from umbilical cord blood by MACS using immunomagnetic beads against CD19 (Miltenyi Biotec) and were used as a positive control for and staining (see Fig. 3).
FIGURE 3. Ig L chain expression resulting from BCR-ABL1 inhibition is heavily biased for L chains. BCR-ABL1+ BV173 cells, NALM1 cells, and SUP-B15 cells were cultured in the presence or absence of 10 μmol/L STI571 for 2 days and surviving cells were analyzed for surface expression of and L chains by flow cytometry. B lymphocytes from umbilical cord blood were enriched by MACS and used as positive control for and staining.
Amplification of double-strand RSS breaks by ligation-mediated PCR
From 2.5 x 106 untreated BV173, NALM1, and SUP-B15 cells or after treatment with 10 μmol/L STI571 after the times indicated, genomic DNA was isolated and ligated to a blunt-end linker using T4 DNA ligase (Invitrogen Life Technologies) at 14°C overnight. The linker was constructed by annealing the oligonucleotides 5'-TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG-3' and 3' amino (C7)-GACGAGCTTAAGTTCGAAGATTGCTACATGCCCCT-5' and protruding 3' overhangs were removed by 3'5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I (Invitrogen Life Technologies). Ligation-mediated PCR (LM-PCR; Ref. 29) was conducted with modifications as previously described (28). In two seminested rounds of amplification (35 and 45 PCR cycles at an annealing temperature of 59°C), RSS intermediates with a DNA double-strand break at the 5' heptamer of J gene segments were amplified (see Figs. 4 and 5A) using 5'-GTAATTAACATTCAGTCTACTTTC-3' as external forward and 5'-TAACATTCAGTCTACTTTCTAAAA-3' as internal forward primers together with 5'-TCCCCGTACATCGTTAGAAG-3' as reverse primer specific for DNA-ligated linker molecules. To amplify RSS intermediates with a DNA double-strand break at the 5' heptamer of J gene segments, 5'-TTCTCACTTCTTCCATGGTGAC-3' and 5'-ACTTCTTCCATGGTGACAGTCT-3' were used in two rounds of PCR amplification as described above (see Fig. 6). Accordingly, 5'-TCCTCCTCACTGAGCCTCCCTTGAAT-3' and 5'-CTCACTGAGCCTCCCTTGAATAGTCC-3' were used to amplify RSS intermediates with a DNA double-strand break at the 5' heptamer of the KDE. LM-PCR products were cloned and five randomly picked clones were sequenced showing that indeed J1 RSS-linker ligation products were amplified.
FIGURE 4. Molecular analysis of recombination events within the IGK locus. A, The rearrangement of V to J segments is initiated by DNA double-strand breaks at the heptamer RSS sequence (RSS) immediately flanking the J segment. DNA double-strand breaks result in hairpin formation at the J-break and 5' phosphorylated blunt ends at the RSS break, which can be ligated to a blunt-ended linker molecule (Linker). Using PCR primers specific for sequences upstream of the RSS and the ligated linker sequence, broken-ended J-RSS DNA intermediates can be amplified. B, During primary recombination events, a V gene segment rearranges to a (mostly upstream located) J gene segment. In secondary rearrangements, pre-existing V-J joints are deleted by juxtaposition of more upstream V to a more downstream J element. C, A preformed V-J joint (top) can be inactivated by two types of rearrangement of the KDE. Using an upstream V-RSS of an unrearranged V-gene segment, rearrangement of the KDE results in a large deletion within the locus including downstream unrearranged V-gene segments, the pre-existing V-J joint, unrearranged J-gene segments, and the C gene (bottom, left). In alternative, the KDE can rearrange to an intronic RSS between the cluster of J-gene segments and the C gene. In this case, KDE rearrangement results in a deletion of the C gene and both enhancers (bottom, right).
FIGURE 5. Inhibition of BCR-ABL1 causes extensive rearrangement of and L chain genes including the KDE. Using LM-PCR, we could amplify short-lived J1-RSS DNA-double strand breaks predominantly in leukemia cells treated with STI571 (10 μmol/L) for 8 h, which demonstrates active recombinase activity within the IGK loci (A). We next investigated V-J and V-J gene rearrangements in STI571-treated and -untreated leukemia cells. Comparing IGK and IGL loci, there was no indication of preferential V-J gene rearrangement (B). As a specificity control, V-J and V-J joints were also amplified from BCR-ABL1+ chronic myeloid leukemia cells (K562 cells). To control for amount and integrity of DNA used, a germline C1 fragment of the TCR locus was amplified from K562 cells. Amplifying specific fragments of KDE germline configuration, KDE rearrangement to an RSS within the J intron and KDE rearrangement to a V-associated RSS, we observed that the KDE was mainly in germline configuration in untreated leukemia cells, whereas STI571 treatment induced KDE rearrangement predominantly to the J intron RSS (C).
FIGURE 6. Sequentially ordered recombination events within the IGK, KDE, and IGL loci. BCR-ABL1+ BV173, NALM1, and SUP-B15 cells were cultured in the presence or absence of 10 μmol/L STI571 for the times indicated. Genomic DNA was isolated from 5 x 106 cells. Broken-ended DNA strand breaks were ligated to linker molecules and subjected to two rounds of PCR amplification using primers specific for breaks at J-RSS, KDE-RSS, and J-RSS sites.
Amplification of V-J and V-J gene rearrangements
Genomic DNA from 5 x 106 STI571-treated (24 h) or untreated BV173, NALM1, and SUP-B15 cells was isolated and subjected to two rounds of seminested PCR (35 and 45 cycles) using six V and nine V family-specific primers together with J- and J-specific primers, respectively. Primers and PCR conditions were used as previously described (30).
Analysis of the genomic configuration of the KDE
Three primers (KDE germline: 5'-CTCACTGAGCCTCCCTTGAATAGTCC-3'; JC intron: 5'-CCGCGGTTCTTTCTCGATTGAGTGG-3'; KDE external reverse: 5'-CTTCATAGACCCTTCAGGCACATGC-3') were added to V family-specific primers for amplification of V-KDE rearrangements (V primers and KDE external reverse primer), JC intron RSS-KDE rearrangements (JC intron and KDE external reverse primer), and KDE in germline configuration (KDE germline and KDE external reverse primer). One-microliter aliquots of the first rounds were used in separate second rounds for the six family-specific V primers together with 5' J primers and the internal reverse KDE primer (5'-AGACAGGTCCTCAGAGGTCAGAGC-3'), and one second round with JC intron and KDE internal reverse and another with KDE germline and internal reverse KDE (see Fig. 5). V family-specific primers used are 5'-GACATCCRGWTGACCCAGTCTCCWTC-3' for V1, 5'-CAGWCTCCACTCTCCCTGYCCGTCA-3' for V2, 5'-TTGTGWTGACRCAGTCTCCAGSCACC-3' for V3, 5'-AGACTCCCTGGCTGTGTCTCTGGGC-3' for V4, 5'-CAGTCTCCAGCATTCATGTCAGCGA-3' for V5, and 5'-TTTCAGTCTGTGACTCCAAAGGAGAA-3' for V6. J-specific primers used are 5'-TTGATYTCCASCTTGGTCCCYTGGC-3' for J1 and J2, 5'-TTGATATCCACTTTGGTCCCAGGGC-3' for J3, 5'-TTGATCTCCACCTTGGTCCCTCCGC-3' for J4, and 5'-TTAATCTCCAGTCGTGTCCCTTGGC-3' for J5.
Analysis of secondary IGK gene rearrangement
To analyze potential secondary IGK recombination events, we tested whether rearrangement of individual J-gene segments occurs sequentially. Genomic DNA were isolated from untreated BCR-ABL1+ pre-B ALL cells or treated with STI571 for the times indicated and subjected to two rounds of seminested PCR. 5'-GCTCAGCTCCTGGGGCTCCTGC-3' and 5'-GACATCCRGWTGACCCAGTCTCCWTC-3' were used as primers specific for the leader and framework region I, respectively, of V1 gene segments. 5'-TTGATYTCCASCTTGGTCCCYTGGC-3' was used for amplification of J1 and J2 gene rearrangements and 5'-TTAATCTCCAGTCGTGTCCCTTGGC-3' for J5.
Results and Discussion
Regulation of genes implicated in Ig L chain expression in BCR-ABL1+ leukemia cells
Comparing genome-wide gene expression profiles from normal bone marrow PBC with two cases of BCR-ABL1+ pre-B ALL generated by the SAGE technique, we identified 16,786 individual transcripts (unique tags) in PBC (26) and each 9,200 in the pre-B ALL cases, respectively. To elucidate transcriptional regulation during the pre-B to immature B cell transition, we identified 182 genes for which a specific role in early B cell development is known (26). Among these genes, transcription factors such as PAX5, E2A, and EBF and genes coding for (pre-) B cell receptor signaling molecules (e.g., LYN, BTK, SYK, FYN) are silenced in the leukemia cells (24). Furthermore, we focused on a subset of 30 genes which are specifically implicated in Ig L chain regulation (Fig. 1). Here, transcription factors that positively regulate Ig L chain gene transcription (IRF4, SPIB, OBF1, IRF8, E2A, AP4, PU.1, CREM, MEF2, ATF1, JUN) are down-regulated in the leukemia cells compared with normal PBC, which argues for a differentiation block at the PBC to immature B cell transition. However, besides genes implicated in Ig L chain gene rearrangement (pre-B to immature B cell transition), also genes coding for components of the pre-B cell receptor (μ-chain: 565 tags in PBC and 48 and 148 tags in leukemia cells (two cases in Fig. 1); VpreB: 278 tags in PBC and 4 and 12 tags in leukemia cells; 5: 1,932 tags in PBC and 12 and 20 tags in leukemia cells) at the pro-B to pre-B cell transition are down-regulated in the leukemia cells. This may suggest that in the leukemia cells, expression and function of the pre-B cell receptor is impaired in general, including its specific function at the PBC to immature B cell checkpoint (2). We next investigated whether down-regulation of the Ig L chain-associated transcription factors IRF4 and SPIB is indeed linked to BCR-ABL1 kinase activity. To this end, we analyzed primary BCR-ABL1+ leukemia samples, which were derived from three patients before and during treatment with the BCR-ABL1-inhibitor STI571 (cases III, IV, V, Ref. 24 , Fig. 1B). Comparing matched pairs of patient-derived leukemia cells, IRF4 and SPIB mRNA levels were increased when BCR-ABL1 kinase activity was blocked by STI571.
Before rearrangement of Ig L chain genes can be initiated, IGK and IGL loci have to be opened to become accessible to the recombination machinery. Therefore, C and C germline transcription indicating transcriptional activation of Ig L chain loci was analyzed by RT-PCR in BCR-ABL1+ cell lines treated with or without STI571 (Fig. 2A). Upon inhibition of BCR-ABL1, germline transcription of C was up-regulated already after 1 h, followed by C germline transcription after 6 h. However, low germline transcription activity at both loci could also be detected in the absence of STI571. These data suggest that IGK and IGL L chain loci become in sequential order accessible to Ig-specific transcription factors and the recombination machinery.
As constituents of the recombination machinery, the expression of recombination activation genes, RAG1 and RAG2, was analyzed by RT-PCR (Fig. 2A). mRNA levels for both RAG genes were significantly up-regulated after 1 h of STI571 treatment. To investigate RAG1 expression in individual cells, RAG1 protein was stained by immunofluorescence in a leukemia cell line (SUP-B15), from which we previously amplified a productive VH3.53-DH2.8-JH6 Ig H chain gene rearrangement (24), in the presence or absence of the BCR-ABL1-inhibitor STI571. Although some cells already exhibit nuclear RAG1 expression before STI571 treatment, RAG1 protein levels were visibly up-regulated after 2 h in some and after 4 h in almost all cells (Fig. 2B).
To determine whether up-regulation of Ig-L chain-associated transcription factors IRF4 and SPIB increased expression of RAG1 and RAG2 and whether C and C germline transcription is followed by the expression of conventional L chains, we analyzed three BCR-ABL1+ pre-B ALL cell lines for and L chain expression in the presence or absence of STI571 by flow cytometry (Fig. 3). After 48 h of STI571 treatment, >50% of the cells had already undergone apoptosis and were excluded from the analysis. Apoptotic or dead cells were identified by annexin V membrane expression and uptake of propidium iodide. Among the surviving cells, 10% exhibit de novo Ig L chain expression (Fig. 3). Unexpectedly, BCR-ABL1 inhibition resulted in a pattern of L chain expression heavily biased for L chains (Fig. 3). Only a few L chain-producing leukemia cells were detectable, while the anti- Ab used clearly identified normal -expressing B cells (Fig. 3). For control stainings, B cells from umbilical cord blood were used that were enriched by MACS for CD19 expression (Fig. 3).
Extensive KDE rearrangement results in preferential L chain expression on STI571-surviving leukemia cells
Using three different PCR strategies (Fig. 4), we investigated at which level the expression of L chains may have been impaired. By LM-PCR (Fig. 4A), we first showed that inhibition of BCR-ABL1 kinase activity induces DNA double-strand breaks at RSS within the IGK locus after 8 h (Fig. 5A). For validation of the LM-PCR assay, PCR products were cloned and five randomly chosen clones were sequenced. Sequence analysis confirmed J1 RSS-linker ligation products (not shown). Such RSS-specific DNA strand breaks represent an initial step within the recombination process of IGK genes and demonstrate that the IGK locus is indeed targeted by the recombination machinery upon treatment with STI571. Because J-RSS-specific DNA strand breaks represent a precondition for V-J gene rearrangements, we next amplified V-J rearrangements from genomic DNA of B cell precursor leukemia cells treated with or without STI571 (Fig. 4B). Although sporadic V-J and V-J joints were already detectable under control conditions, V-J rearrangements at both IGK and IGL loci were clearly inducible by STI571 (Fig. 5B). As a specificity control, genomic DNA of BCR-ABL1+ chronic myeloid leukemia cells was also subjected to amplification of V-J and V-J rearrangements but no product was obtained. Integrity of DNA isolated from chronic myeloid leukemia cells was verified by amplification of a C1 germline fragment of the TCR locus (Fig. 5B). In agreement with a previous study on the effect of STI571 on v-abl-transformed murine pre-B cell lines (21), we observed a polyclonal pattern of V and V gene rearrangement upon STI571 treatment. STI571-induced V-J gene rearrangements involved all six IGK and all nine IGL V gene families. Whether IGK and IGL genes were also rearranged at similar frequencies in response to STI571 treatment remains unclear, because the PCR approach used here was not quantitative.
Rearrangement of the KDE may lead to inactivation of productively recombined V-J joints and hence represents another level at which Ig L chain expression may be impaired (15). To assess whether pre-existing functional V-J joints may have been inactivated by rearrangement of the KDE, we analyzed both types of KDE recombination within the IGK locus (Fig. 4C). The KDE can rearrange either to an RSS site within the intron region between the cluster of J gene segments and the intronic enhancer or to an RSS site immediately flanking a V gene segment (Fig. 4C). KDE rearrangement leads to deletion of both enhancers and the C gene in the former (J intron RSS-KDE) or to deletion of a V-J joint in the latter case (V RSS-KDE). To investigate whether KDE rearrangement may contribute to reduced L chain usage in STI571-treated leukemia cells, we amplified specific DNA fragments for KDE-germline configuration, J intron RSS-KDE rearrangement, and V RSS-KDE rearrangement from three BCR-ABL1+ leukemia cell lines (BV173, NALM1, SUP-B15) in the absence or presence of STI571 (Fig. 5C). Although KDE was found in germline configuration in untreated leukemia cells, STI571-mediated inhibition of BCR-ABL1 induced deletion of IGK alleles by rearrangement of the KDE to J intron RSS sites and to V RSS sites. Targeting of the IGK locus by the recombination machinery and generation of V-J joints appears to be normal, but overrepresentation of L chains can be attributed to extensive KDE rearrangement observed here (Fig. 5C). In the absence of BCR-ABL1 kinase activity, differentiating leukemia cells seem to undergo multiple rounds of rearrangement targeting both and L chain loci, which ultimately leads to preferential L chain expressiondue to KDE-mediated deletion of potentially productive V-J joints.
Rearrangements at the IGK and IGL loci occur in sequential order
For this reason, we considered STI571-induced Ig L chain gene rearrangement as a model for the pre-B to immature B cell transition in normal B cell development and investigated whether IGK, KDE, and IGL gene rearrangements follow a defined sequence of events. Sequential rearrangement of IGK, KDE, and IGL genes would be predicted by the ordered model of L chain gene recombination. To the contrary, according to the stochastic model of L chain gene rearrangement, IGK, KDE, and IGL genes would be recombined independently at each locus. Therefore, we incubated the three BCR-ABL1+ pre-B ALL cell lines in the presence or absence of STI571 and analyzed genomic DNA for RSS-specific double-strand breaks within the IGK, KDE, and IGL loci at different time points (Fig. 6). In the three cell lines, RSS-specific DNA double-strand breaks were already detectable at the IGK locus within 1 h after STI571 treatment. RSS breaks flanking the KDE followed after 6 h of STI571 treatment and RSS-DNA breaks were found at the IGL locus only after 12 h (Fig. 6). These findings are consistent with a sequential order of rearrangement events: ablation of BCR-ABL1 kinase activity almost immediately initiates IGK gene rearrangements, many of which are subsequently deleted by KDE- and ultimately replaced by IGL-gene rearrangements (Fig. 6). Of note, up-regulation of C germline transcripts did not precede the onset of KDE RSS breaks (Figs. 2A and 6). Unlike normal B cell development, only very few STI571-treated leukemia cells seem to stay at a + stage, while the vast majority of cells continue to rearrange, first KDE and ultimately IGL genes. It should be noted that between 12 and 24 h of STI571 incubation, RSS breaks could be detected concomitantly in IGK, KDE, and IGL loci (Fig. 6). Therefore, we cannot exclude that during this period of time, IGL gene rearrangements may have occurred before recombination within the IGK locus. Stability of RSS-strand break intermediates is cell cycle-dependent and mainly found in G1 and G0 phases (29). Indeed, STI571 treatment induces cell cycle arrest in BCR-ABL1+ B cell precursor leukemia cells (25). Thus, the detection of J-RSS breaks for an extended period of time (1–24 h), partially overlapping with KDE-RSS and even J-RSS breaks, can be explained by cell cycle arrest and increased stability of RSS breaks induced by STI571.
Indication for secondary IGK gene rearrangements
In contrast, the relatively wide time frame during which IGK gene rearrangements can occur might reflect that the leukemia cells are not all synchronized with respect to STI571-induced IGK gene rearrangement. This is consistent with a rather heterogenous pattern of RAG1 up-regulation following treatment with STI571 (Fig. 2). Moreover, RSS-specific DNA-strand breaks within the IGK locus do not necessarily reflect a single definitive V-J gene rearrangement and would also arise from any secondary recombination event during L chain revision (Fig. 4B). To determine whether ongoing L chain editing may extend the time window for RSS-specific DNA-strand breaks, we searched for traces of secondary rearrangements within the IGK locus. A pre-existing V-J joint can only be replaced by a rearrangement which uses an upstream-located V- together with a downstream-located J segment (Fig. 4B). Individual V segments are not arranged in the order of V gene families within the IGK locus. Therefore, we only compared the usage of upstream J1 and J2 segments to downstream J5 elements (see Fig. 4B) at different time points after STI571-induced initiation of L chain gene rearrangement. Consistent with a shift from upstream to downstream J elements, J1 and J2 gene rearrangements were detected slightly earlier than rearrangements involving the J5 gene segment (Fig. 7). Given that the two round PCR approach used here for analysis of V-J gene rearrangements is not quantitative, the relative amount of PCR products does not necessarily reflect the usage of individual J segments.
FIGURE 7. Shifting the preferential J usage from upstream to downstream located J gene segments indicates secondary IGK gene rearrangements. V1 gene rearrangements were amplified from three BCR-ABL1+ pre-B ALL cell lines using V1 leader- and V1 framework region I-specific primers together with either primers matching to J1 and J2 or primers matching to J5. As controls, PCRs were performed to which water was added instead of DNA.
Concluding remarks
We conclude that inhibition of BCR-ABL1 initiates a coordinated sequence of Ig L chain gene rearrangement events: almost immediately after ablation of BCR-ABL1 kinase activity by STI571, the IGK locus opens and is targeted by the recombination machinery. The continuous generation of new J RSS-specific DNA strand breaks even after 24 h together with a shift from upstream J1 and J2 elements to downstream J5 segments suggests that initial V-J rearrangements are followed by at least one, perhaps multiple, round(s) of secondary rearrangements. A possible initiation signal for subsequent KDE rearrangement could be exhaustion of the recombination potential of IGK alleles in multiple rounds of V-J rearrangement. Recombination of the KDE deletes V-J rearrangements from the chromosome and may initiate V-J gene rearrangements, which can first be detected after 12 h. According to these observations, it is not surprising that the vast majority of leukemia subclones that have been primed to differentiate, ultimately express L chains on their surface (Fig. 3). Obviously, the leukemia cells, unlike normal PBC, are lacking a feedback mechanism which prompts them to halt the recombination machinery in the presence of a productively rearranged IGK allele. Upon inhibition of BCR-ABL1, the leukemia cells seem to enter a pre-determined (ordered) sequence of rearrangement events, in which -expressing cells would only exist as a transition stage, inevitably leading to the expression of L chains.
Acknowledgments
We thank Stefanie Jauch and Peter Wurst for excellent technical assistance, Mario Fabri, Hassan Jumaa, Klaus Rajewsky, and Michael Reth for critical discussions, Martin Kr?nke and Janet D. Rowley for continuous support and discussions, and Sean McCleary for proofreading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Deutsche Forschungsgemeinschaft through Grants MU1616/2-1 and MU1616/3-1 (to M.M.), the German José-Carreras-Leukemia-Foundation (grant to M.M.), and the Ministry of Science and Research for North Rhine-Westphalia through the Stem Cell Network North-Rhine-Westphalia (to M.M.). F.K. is supported by scholarships of the Studienstiftung des deutschen Volkes and the K?ln Fortune program of the Faculty of Medicine of the University of Cologne. M.M. is supported by the Deutsche Forschungsgemeinschaft through the Emmy-Noether-Program.
2 Address correspondence and reprint requests to Dr. Markus Müschen, Laboratory for Molecular Stem Cell Biology, Building 14.80, Heinrich-Heine-Universit?t Düsseldorf, 40225 Düsseldorf, Germany. E-mail address: markus.mueschen@uni-duesseldorf.de
3 Abbreviations used in this paper: IGH, Ig μ H chain; PBC, pre-B cell; IGK, Ig L chain; IGL, Ig L chain; KDE, -deleting element; RSS, recombination signal sequence; ALL, acute lymphoblastic leukemia; SAGE, serial analysis of gene expression; HSC, hemopoietic progenitor cell; CMP, common myeloid progenitor cell; NBC, naive B cell; GCB, germinal center B cell; MBC, memory B cell; PC, plasma cell; TLP, T lymphoid progenitor; LM-PCR, ligation-mediated PCR.
Received for publication March 4, 2004. Accepted for publication October 18, 2004.
References
Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.
Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature 381:751.
Alt, F. W., G. D. Yancopoulos, T. K. Blackwell, C. Wood, E. Thomas, M. Boss, R. Coffman, N. Rosenberg, S. Tonegawa, D. Baltimore. 1984. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 3:1209.
Langman, R. E., M. Cohn. 1995. The proportion of B-cell subsets expressing and light chains changes following antigenic selection. Immunol. Today 16:141
Nadel, B., P. A. Cazenave, P. Sanchez. 1990. Murine gene rearrangements: the stochastic model prevails over the ordered model. EMBO J. 9:435.
Arakawa, H., T. Shimizu, S. Takeda. 1996. Re-evaluation of the probabilities for productive arrangements on the and loci. Int. Immunol. 8:91.
Gorman, J. R., N. van der Stoep, R. Monroe, M. Cogne, L. Davidson, F. W. Alt. 1996. The Ig enhancer influences the ratio of Ig versus Ig B lymphocytes. Immunity 5:241.
Inlay, M., F. W. Alt, D. Baltimore, Y. Xu. 2002. Essential roles of the light chain intronic enhancer and 3' enhancer in rearrangement and demethylation. Nat. Immunol. 3:463.
Zou, Y. R., S. Takeda, K. Rajewsky. 1993. Gene targeting in the Ig locus: efficient generation of chain-expressing B cells, independent of gene rearrangements in Ig . EMBO J. 12:811
Chen, J., M. Trounstine, C. Kurahara, F. Young, C. C. Kuo, Y. Xu, J. F. Loring, F. W. Alt, D. Huszar. 1993. B cell development in mice that lack one or both immunoglobulin light chain genes. EMBO J. 12:821.
Korsmeyer, S. J., P. A. Hieter, S. O. Sharrow, C. K. Goldman, P. Leder, T. A. Waldmann. 1982. Normal human B cells display ordered light chain gene rearrangements and deletions. J. Exp. Med. 156:975.
Korsmeyer, S. J., P. A. Hieter, J. V. Ravetch, D. G. Poplack, T. A. Waldmann, P. Leder. 1981. Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc. Natl. Acad. Sci. USA 78:7096.
Look, A.T.. 1997. Oncogenic transcription factors in the human acute leukemias. Science 278:1059.
Smith, K. M., R. Yacobi, R. A. Van Etten. 2003. Autoinhibition of Bcr-Abl through its SH3 domain. Mol. Cell. 12:27
LeBien, T. W.. 2000. Fates of human B-cell precursors. Blood 96:9.
Chen, Y. Y., L. C. Wang, M. S. Huang, N. Rosenberg. 1994. An active v-abl protein tyrosine kinase blocks immunoglobulin light-chain gene rearrangement. Genes Dev. 8:688.
Muljo, S. A., M. S. Schlissel. 2003. A small molecule Abl kinase inhibitor induces differentiation of Abelson virus-transformed pre-B cell lines. Nat. Immunol. 4:31.
Druker, B. J., C. L. Sawyers, H. Kantarjian, D. J. Resta, S. F. Reese, J. M. Ford, R. Capdeville, M. Talpaz. 2001. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:1038.
Hofmann, W. K., S. de Vos, D. Elashoff, H. Gschaidmeier, D. Hoelzer, H. P. Koeffler, O. G. Ottmann. 2002. Relation between resistance of Philadelphia-chromosome-positive acute lymphoblastic leukaemia to the tyrosine kinase inhibitor STI571 and gene-expression profiles: a gene-expression study. Lancet 359:481.
Klein, F., N. Feldhahn, L. Harder, H. Wang, M. Wartenberg, W. K. Hofmann, P. Wernet, R. Siebert, M. Müschen. 2004. The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells. J. Exp. Med. 199:673.
Deininger, M. W., S. A. Vieira, Y. Parada, L. Banerji, E. W. Lam, G. Peters, F. X. Mahon, T. Kohler, J. M. Goldman, J. V. Melo. 2001. Direct relation between BCR-ABL tyrosine kinase activity and cyclin D2 expression in lymphoblasts. Cancer Res. 61:8005
Müschen, M., S. Lee, G. Zhou, N. Feldhahn, V. S. Barath, J. Chen, C. Moers, M. Kr?nke, J. D. Rowley, S. M. Wang. 2002. Molecular portraits of B cell lineage commitment. Proc. Natl. Acad. Sci.(Florian Klein, Niklas Fel)
The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, C and C germline transcription, and rearrangement of Ig L chain (IGK) and Ig L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed L, but almost no L chains. This could be explained by STI571-induced rearrangement of the -deleting element (KDE), which can delete productively rearranged V-J joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of V-J joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of C and C germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located J- to downstream J-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.
Introduction
B cell precursors within the bone marrow undergo a sequence of Ig gene rearrangements defining distinct stages of early B cell differentiation (1). During their early development, B cell precursors have to pass checkpoints at which only cells carrying functional Ig gene rearrangements are selected for further development along the B cell lineage (2). For instance, the presence of a productive Ig μ H chain (IGH)3 gene rearrangement is a prerequisite for the expression of the Ig μ H chain as a component of the pre-B cell receptor on the surface of pre-B cells (PBC). As soon as a B cell precursor successfully rearranges VH, DH, and JH gene segments on one allele, the recombination machinery is halted. This prevents further rearrangement of the second allele, which constitutes allelic exclusion at the IGH locus (3). Termination of the rearrangement process at the IGH locus is followed by the initiation of Ig L chain gene recombination at the Ig L chain (IGK) and Ig L chain (IGL) loci, which defines the pre-B to immature B cell transition. Human B cells express more frequently L than L chains, at a / ratio of 1.4 (4). To explain the relative overrepresentation of IGK gene rearrangements, which is even more striking in mice, two models have been proposed: the "stochastic model" (5, 6) postulates that IGK and IGL loci are independently accessible to recombinase activity and that cis-acting regulatory elements (e.g., the 3' enhancer; Refs. 7 and 8) determine the ratio of L and L chain-expressing cells. This model was supported by the finding that -expressing B cells could arise from mice carrying inactivated IGK loci on both alleles (9, 10). On the contrary, the "ordered model" proposes that IGK genes, as a rule, rearrange before IGL gene recombination (11, 12). Consistent with an ordered model of IGK and IGL gene rearrangement, recent work showed that single human -expressing B cells (13) or B cell precursor leukemia clones (14) harbored inactivated V-J gene rearrangements almost in all instances in addition to productive V-J joints but not vice versa: only few -expressing B cells or leukemic B cell clones also carried inactive V-J joints in addition to productive V-J gene rearrangements. These findings argue for sequential rearrangement of IGK and IGL genes. However, these studies do not formally demonstrate that rearrangement of IGK genes in -expressing B cells do necessarily precede successful V-J gene recombination. In -expressing B cells, for instance, IGL genes may well be rearranged first, followed by needless V-J gene rearrangements, which then would subsequently be inactivated by rearrangement of the -deleting element (KDE). The KDE may rearrange to recombination signal sequences (RSS) flanking germline V gene segments or an RSS within the intron between the J cluster and the C gene, which leads to inactivation of a pre-existing V-J joint in either case (15). Also the existence of B cells expressing L together with L chains (16) suggests that recombination events in the IGK and IGL loci are not mutually exclusive in all instances. Instead of L or L chains, human PBC express surrogate L chains composed of VpreB and 5.
In the vast majority of acute lymphoblastic leukemia (ALL), the malignant clone represents the outgrowth of a transformed PBC. In many cases, pre-B ALL clones carry specific oncogenic gene rearrangements defining both biological and clinical subentities (17). Among these translocation events, the t(9;22)(q34;q11) results in a fusion of the BCR and ABL1 genes, which codes for a constitutive active variant of the ABL1 tyrosine kinase (18) and represents the most frequent recurrent aberration leading to ALL in adults (19). ABL1 represents the human homologue of the transforming gene of the Abelson murine leukemia virus, v-abl. Although v-abl has been used for many years to transform murine PBC, recent work showed that v-abl also confers a differentiation block at the PBC stage of development and, hence, prevents rearrangement of IGK or IGL L chain genes (20, 21). Recently, a specifically designed inhibitor of BCR-ABL1, termed STI571, has become available and is now widely used as an anti-leukemia drug for BCR-ABL1+ leukemias (22). Therefore, we investigated whether and in which way inhibition of BCR-ABL1 kinase activity by STI571 might induce differentiation including IGK and IGL gene rearrangement in pre-B ALL cells.
Materials and Methods
Patient samples, primary cells, and cell lines
BCR-ABL1+ pre-B ALL cells from five patients were analyzed. From three of these patients, leukemia cells were available before and during treatment with STI571 (cases III, IV, and V, Refs. 23 and 24). Samples of two other cases (cases I and II, Ref. 24) were used for serial analysis of gene expression (SAGE) analysis. Normal human CD10+VpreB+ PBC, CD34+CD38lowCD133+ hemopoietic progenitor cells (HSC),CD15+CD34– myeloid progenitor cells, CD7+CD10+ T lymphoid progenitor cells (TLP) and mature B cell subsets including CD19+CD27– naive B cells (NBC), CD20+CD77+ germinal center B cells (GCB), CD19+CD27+ memory B cells (MBC), and CD19lowCD138+ plasma cells (PC) were isolated by MACS and FACS from bone marrow, umbilical cord blood, peripheral blood, or tonsils from healthy donors as previously described (25, 26, 27, 28). For cell culture experiments, the BCR-ABL1+ pre-B ALL cell lines BV173, NALM1, and SUP-B15 (Deutsche Sammlung von Mikroorganismen und Zellkulturen) were used. Cell lines were treated with 10 μmol/L STI571 (Novartis) for the times indicated.
SAGE analysis
To identify differentially expressed genes between BCR-ABL1+ pre-B ALL cells and normal PBC that play a role in Ig L chain gene regulation, we analyzed mRNA expression profiles generated by the SAGE method. A total of 592,000 SAGE tags were collected for 10 SAGE profiles. A total of 106,000 tags were analyzed from the CD34+ HSC library, 99,000 for CD15+ common myeloid progenitor cells (CMP), 110,000 for CD10+CD19+ PBC, 96,500 for CD7+CD10+ TLP, and each 30,000 tags for two cases of bone marrow-derived pre-B ALL carrying a BCR-ABL1 gene rearrangement (cases I and II), CD19+CD27– NBC, CD20+ CD77+ GCB, CD19+CD27+ MBC, and CD19+CD138+ PC (see Fig. 1A). All SAGE libraries were normalized to 100,000 tags. SAGE data were graphically visualized using the Cluster and Treeview software (http://rana.lbl.gov/) and sorted according to the ratio between SAGE-tag counts in PBC and in BCR-ABL1+ ALL cases (26, 27, 28). In these SAGE profiles, 30 genes were identified which have been implicated in the regulation of Ig L chain expression. These genes were searched in the literature, as well as in UniGene (
FIGURE 1. mRNA levels of Ig L chain-associated genes in BCR-ABL1+ pre-B ALL cells. A, Focusing on 30 genes implicated in the regulation of Ig L chain expression, genome-wide SAGE profiles of two BCR-ABL1+ pre-B ALL cases (ALL1 and ALL2) were compared with SAGE profiles of normal CD34+CD38low HSC, CD15+ CMP, CD7+CD10+ TLP, CD10+CD19+ PBC, CD19+CD27– NBC, CD20+CD77+ GCB, CD19+CD27+ MBC, and CD19lowCD138+ PC. SAGE-tag counts are visualized by colors, red indicating high and green depicting low levels or no expression, respectively. SAGE data were sorted according to the ratio of SAGE-tag counts in normal B cell subsets to leukemia samples. B, From three patients with BCR-ABL1+ pre-B ALL, leukemia samples were available before and during treatment with the BCR-ABL1 kinase inhibitor STI571. In these matched leukemia sample pairs, mRNA levels of IRF4 and SPIB were compared by semiquantitative RT-PCR. cDNA amounts were normalized by OD measurements and amplification of a specific fragment of the housekeeping gene GAPDH.
RT-PCR analysis of IRF4, SPIB, and RAG1 and RAG2 expression and germline C and C transcripts
mRNA levels of the Ig L chain-associated transcription factors IRF4 and SPIB were measured in BCR-ABL1+ leukemia samples from three patients (cases III, IV, and V, Ref. 24) before and during treatment with STI571 (see Fig. 1). From patient samples, total RNA was isolated and transcribed into cDNA as previously described (26, 27, 28). Amounts of cDNA were normalized by OD measurement and semiquantitative RT-PCR using 5'-TTAGCACCCCTGGCCAAG-3' and 5'-CTTACTCCTTGGAGGCCATG-3' for amplification of a cDNA fragment of GAPDH. For semiquantitative RT-PCR analysis of IRF4 the oligonucleotides 5'-CAAGAGCAATGACTTTGAGG-3' and 5'-TGGGACATTGGTACGGGAT-3' were used and for SPIB 5'-AAGACTTACCGTTGGACAGC-3' and 5'-CTTGGAGGAGAACTGGAAGA-3'.
To determine L chain locus germline transcription by RT-PCR, RNA was isolated from BV173, NALM1, and SUP-B15 cell lines with or without STI571 incubation, reverse-transcribed into cDNA, and normalized as described above. For amplification of C germline transcripts, 5'-TTCAACAGGGGAGAGTGTTAGAG-3' and 5'-ATGCGCCTTAGGATGACTACATA-3' primers were used, and 5'-TGTCTGATCAGTGACTTCTACCC-3' and 5'-CTGTAGCTTCTGTGGGACTTC-3' primers were used for C germline transcripts. RT-PCR for RAG1 and RAG2 was conducted using the oligonucleotides 5'-ATAGAAGAAAGCAACACAAAAGC-3' and 5'-ATACTGAGTTCAATCCCTGAAGA-3' for RAG1 and 5'-ATAGCAAGAGCTCTACACACTCC-3' and 5'-AAAAATCAGATCAGAAATCCTCA-3' for RAG2 (see Fig. 2A).
FIGURE 2. Inhibition of BCR-ABL1 by STI571 results in C and C germline transcription and rapid up-regulation of RAG1 and RAG2. Three BCR-ABL1+ pre-B ALL cell lines were incubated in the presence or absence of 10 μmol/L STI571 for the times indicated and analyzed for the expression of C and C germline transcription, RAG1, RAG2, and GAPDH (control) by RT-PCR (A). Nuclear RAG1 expression in individual leukemia cells was measured by staining for CD19 (green) and intracellular RAG1 (red; B).
Analysis of nuclear RAG1 expression
BCR-ABL1+ pre-B ALL cells (BV173 and SUP-B15) were cultured in the presence or absence of STI571 for 1, 2, or 4 h and subsequently stained with a Cy5-labeled Ab against CD19. After treatment with methanol and 4% paraformaldehyde, cells were stained with a mouse anti-human RAG1 IgG1 (BD Biosciences) together with a Cy2-labeled goat anti-mouse IgG Ab. Expression of CD19 and RAG1 was visualized by immunofluorescence (see Fig. 2B).
Flow cytometry
Surface expression of IGK or IGL on BV173, NALM1, and SUP-B15 pre-B ALL cells in the presence or absence of STI571 was monitored using Abs against IGK or IGL (BD Biosciences) after the incubation times indicated (see Fig. 3). Apoptotic or dead cells were identified by staining with FITC-labeled annexin V and propidium iodide (BD Biosciences) and excluded from analysis. Normal B cells were enriched from umbilical cord blood by MACS using immunomagnetic beads against CD19 (Miltenyi Biotec) and were used as a positive control for and staining (see Fig. 3).
FIGURE 3. Ig L chain expression resulting from BCR-ABL1 inhibition is heavily biased for L chains. BCR-ABL1+ BV173 cells, NALM1 cells, and SUP-B15 cells were cultured in the presence or absence of 10 μmol/L STI571 for 2 days and surviving cells were analyzed for surface expression of and L chains by flow cytometry. B lymphocytes from umbilical cord blood were enriched by MACS and used as positive control for and staining.
Amplification of double-strand RSS breaks by ligation-mediated PCR
From 2.5 x 106 untreated BV173, NALM1, and SUP-B15 cells or after treatment with 10 μmol/L STI571 after the times indicated, genomic DNA was isolated and ligated to a blunt-end linker using T4 DNA ligase (Invitrogen Life Technologies) at 14°C overnight. The linker was constructed by annealing the oligonucleotides 5'-TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG-3' and 3' amino (C7)-GACGAGCTTAAGTTCGAAGATTGCTACATGCCCCT-5' and protruding 3' overhangs were removed by 3'5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I (Invitrogen Life Technologies). Ligation-mediated PCR (LM-PCR; Ref. 29) was conducted with modifications as previously described (28). In two seminested rounds of amplification (35 and 45 PCR cycles at an annealing temperature of 59°C), RSS intermediates with a DNA double-strand break at the 5' heptamer of J gene segments were amplified (see Figs. 4 and 5A) using 5'-GTAATTAACATTCAGTCTACTTTC-3' as external forward and 5'-TAACATTCAGTCTACTTTCTAAAA-3' as internal forward primers together with 5'-TCCCCGTACATCGTTAGAAG-3' as reverse primer specific for DNA-ligated linker molecules. To amplify RSS intermediates with a DNA double-strand break at the 5' heptamer of J gene segments, 5'-TTCTCACTTCTTCCATGGTGAC-3' and 5'-ACTTCTTCCATGGTGACAGTCT-3' were used in two rounds of PCR amplification as described above (see Fig. 6). Accordingly, 5'-TCCTCCTCACTGAGCCTCCCTTGAAT-3' and 5'-CTCACTGAGCCTCCCTTGAATAGTCC-3' were used to amplify RSS intermediates with a DNA double-strand break at the 5' heptamer of the KDE. LM-PCR products were cloned and five randomly picked clones were sequenced showing that indeed J1 RSS-linker ligation products were amplified.
FIGURE 4. Molecular analysis of recombination events within the IGK locus. A, The rearrangement of V to J segments is initiated by DNA double-strand breaks at the heptamer RSS sequence (RSS) immediately flanking the J segment. DNA double-strand breaks result in hairpin formation at the J-break and 5' phosphorylated blunt ends at the RSS break, which can be ligated to a blunt-ended linker molecule (Linker). Using PCR primers specific for sequences upstream of the RSS and the ligated linker sequence, broken-ended J-RSS DNA intermediates can be amplified. B, During primary recombination events, a V gene segment rearranges to a (mostly upstream located) J gene segment. In secondary rearrangements, pre-existing V-J joints are deleted by juxtaposition of more upstream V to a more downstream J element. C, A preformed V-J joint (top) can be inactivated by two types of rearrangement of the KDE. Using an upstream V-RSS of an unrearranged V-gene segment, rearrangement of the KDE results in a large deletion within the locus including downstream unrearranged V-gene segments, the pre-existing V-J joint, unrearranged J-gene segments, and the C gene (bottom, left). In alternative, the KDE can rearrange to an intronic RSS between the cluster of J-gene segments and the C gene. In this case, KDE rearrangement results in a deletion of the C gene and both enhancers (bottom, right).
FIGURE 5. Inhibition of BCR-ABL1 causes extensive rearrangement of and L chain genes including the KDE. Using LM-PCR, we could amplify short-lived J1-RSS DNA-double strand breaks predominantly in leukemia cells treated with STI571 (10 μmol/L) for 8 h, which demonstrates active recombinase activity within the IGK loci (A). We next investigated V-J and V-J gene rearrangements in STI571-treated and -untreated leukemia cells. Comparing IGK and IGL loci, there was no indication of preferential V-J gene rearrangement (B). As a specificity control, V-J and V-J joints were also amplified from BCR-ABL1+ chronic myeloid leukemia cells (K562 cells). To control for amount and integrity of DNA used, a germline C1 fragment of the TCR locus was amplified from K562 cells. Amplifying specific fragments of KDE germline configuration, KDE rearrangement to an RSS within the J intron and KDE rearrangement to a V-associated RSS, we observed that the KDE was mainly in germline configuration in untreated leukemia cells, whereas STI571 treatment induced KDE rearrangement predominantly to the J intron RSS (C).
FIGURE 6. Sequentially ordered recombination events within the IGK, KDE, and IGL loci. BCR-ABL1+ BV173, NALM1, and SUP-B15 cells were cultured in the presence or absence of 10 μmol/L STI571 for the times indicated. Genomic DNA was isolated from 5 x 106 cells. Broken-ended DNA strand breaks were ligated to linker molecules and subjected to two rounds of PCR amplification using primers specific for breaks at J-RSS, KDE-RSS, and J-RSS sites.
Amplification of V-J and V-J gene rearrangements
Genomic DNA from 5 x 106 STI571-treated (24 h) or untreated BV173, NALM1, and SUP-B15 cells was isolated and subjected to two rounds of seminested PCR (35 and 45 cycles) using six V and nine V family-specific primers together with J- and J-specific primers, respectively. Primers and PCR conditions were used as previously described (30).
Analysis of the genomic configuration of the KDE
Three primers (KDE germline: 5'-CTCACTGAGCCTCCCTTGAATAGTCC-3'; JC intron: 5'-CCGCGGTTCTTTCTCGATTGAGTGG-3'; KDE external reverse: 5'-CTTCATAGACCCTTCAGGCACATGC-3') were added to V family-specific primers for amplification of V-KDE rearrangements (V primers and KDE external reverse primer), JC intron RSS-KDE rearrangements (JC intron and KDE external reverse primer), and KDE in germline configuration (KDE germline and KDE external reverse primer). One-microliter aliquots of the first rounds were used in separate second rounds for the six family-specific V primers together with 5' J primers and the internal reverse KDE primer (5'-AGACAGGTCCTCAGAGGTCAGAGC-3'), and one second round with JC intron and KDE internal reverse and another with KDE germline and internal reverse KDE (see Fig. 5). V family-specific primers used are 5'-GACATCCRGWTGACCCAGTCTCCWTC-3' for V1, 5'-CAGWCTCCACTCTCCCTGYCCGTCA-3' for V2, 5'-TTGTGWTGACRCAGTCTCCAGSCACC-3' for V3, 5'-AGACTCCCTGGCTGTGTCTCTGGGC-3' for V4, 5'-CAGTCTCCAGCATTCATGTCAGCGA-3' for V5, and 5'-TTTCAGTCTGTGACTCCAAAGGAGAA-3' for V6. J-specific primers used are 5'-TTGATYTCCASCTTGGTCCCYTGGC-3' for J1 and J2, 5'-TTGATATCCACTTTGGTCCCAGGGC-3' for J3, 5'-TTGATCTCCACCTTGGTCCCTCCGC-3' for J4, and 5'-TTAATCTCCAGTCGTGTCCCTTGGC-3' for J5.
Analysis of secondary IGK gene rearrangement
To analyze potential secondary IGK recombination events, we tested whether rearrangement of individual J-gene segments occurs sequentially. Genomic DNA were isolated from untreated BCR-ABL1+ pre-B ALL cells or treated with STI571 for the times indicated and subjected to two rounds of seminested PCR. 5'-GCTCAGCTCCTGGGGCTCCTGC-3' and 5'-GACATCCRGWTGACCCAGTCTCCWTC-3' were used as primers specific for the leader and framework region I, respectively, of V1 gene segments. 5'-TTGATYTCCASCTTGGTCCCYTGGC-3' was used for amplification of J1 and J2 gene rearrangements and 5'-TTAATCTCCAGTCGTGTCCCTTGGC-3' for J5.
Results and Discussion
Regulation of genes implicated in Ig L chain expression in BCR-ABL1+ leukemia cells
Comparing genome-wide gene expression profiles from normal bone marrow PBC with two cases of BCR-ABL1+ pre-B ALL generated by the SAGE technique, we identified 16,786 individual transcripts (unique tags) in PBC (26) and each 9,200 in the pre-B ALL cases, respectively. To elucidate transcriptional regulation during the pre-B to immature B cell transition, we identified 182 genes for which a specific role in early B cell development is known (26). Among these genes, transcription factors such as PAX5, E2A, and EBF and genes coding for (pre-) B cell receptor signaling molecules (e.g., LYN, BTK, SYK, FYN) are silenced in the leukemia cells (24). Furthermore, we focused on a subset of 30 genes which are specifically implicated in Ig L chain regulation (Fig. 1). Here, transcription factors that positively regulate Ig L chain gene transcription (IRF4, SPIB, OBF1, IRF8, E2A, AP4, PU.1, CREM, MEF2, ATF1, JUN) are down-regulated in the leukemia cells compared with normal PBC, which argues for a differentiation block at the PBC to immature B cell transition. However, besides genes implicated in Ig L chain gene rearrangement (pre-B to immature B cell transition), also genes coding for components of the pre-B cell receptor (μ-chain: 565 tags in PBC and 48 and 148 tags in leukemia cells (two cases in Fig. 1); VpreB: 278 tags in PBC and 4 and 12 tags in leukemia cells; 5: 1,932 tags in PBC and 12 and 20 tags in leukemia cells) at the pro-B to pre-B cell transition are down-regulated in the leukemia cells. This may suggest that in the leukemia cells, expression and function of the pre-B cell receptor is impaired in general, including its specific function at the PBC to immature B cell checkpoint (2). We next investigated whether down-regulation of the Ig L chain-associated transcription factors IRF4 and SPIB is indeed linked to BCR-ABL1 kinase activity. To this end, we analyzed primary BCR-ABL1+ leukemia samples, which were derived from three patients before and during treatment with the BCR-ABL1-inhibitor STI571 (cases III, IV, V, Ref. 24 , Fig. 1B). Comparing matched pairs of patient-derived leukemia cells, IRF4 and SPIB mRNA levels were increased when BCR-ABL1 kinase activity was blocked by STI571.
Before rearrangement of Ig L chain genes can be initiated, IGK and IGL loci have to be opened to become accessible to the recombination machinery. Therefore, C and C germline transcription indicating transcriptional activation of Ig L chain loci was analyzed by RT-PCR in BCR-ABL1+ cell lines treated with or without STI571 (Fig. 2A). Upon inhibition of BCR-ABL1, germline transcription of C was up-regulated already after 1 h, followed by C germline transcription after 6 h. However, low germline transcription activity at both loci could also be detected in the absence of STI571. These data suggest that IGK and IGL L chain loci become in sequential order accessible to Ig-specific transcription factors and the recombination machinery.
As constituents of the recombination machinery, the expression of recombination activation genes, RAG1 and RAG2, was analyzed by RT-PCR (Fig. 2A). mRNA levels for both RAG genes were significantly up-regulated after 1 h of STI571 treatment. To investigate RAG1 expression in individual cells, RAG1 protein was stained by immunofluorescence in a leukemia cell line (SUP-B15), from which we previously amplified a productive VH3.53-DH2.8-JH6 Ig H chain gene rearrangement (24), in the presence or absence of the BCR-ABL1-inhibitor STI571. Although some cells already exhibit nuclear RAG1 expression before STI571 treatment, RAG1 protein levels were visibly up-regulated after 2 h in some and after 4 h in almost all cells (Fig. 2B).
To determine whether up-regulation of Ig-L chain-associated transcription factors IRF4 and SPIB increased expression of RAG1 and RAG2 and whether C and C germline transcription is followed by the expression of conventional L chains, we analyzed three BCR-ABL1+ pre-B ALL cell lines for and L chain expression in the presence or absence of STI571 by flow cytometry (Fig. 3). After 48 h of STI571 treatment, >50% of the cells had already undergone apoptosis and were excluded from the analysis. Apoptotic or dead cells were identified by annexin V membrane expression and uptake of propidium iodide. Among the surviving cells, 10% exhibit de novo Ig L chain expression (Fig. 3). Unexpectedly, BCR-ABL1 inhibition resulted in a pattern of L chain expression heavily biased for L chains (Fig. 3). Only a few L chain-producing leukemia cells were detectable, while the anti- Ab used clearly identified normal -expressing B cells (Fig. 3). For control stainings, B cells from umbilical cord blood were used that were enriched by MACS for CD19 expression (Fig. 3).
Extensive KDE rearrangement results in preferential L chain expression on STI571-surviving leukemia cells
Using three different PCR strategies (Fig. 4), we investigated at which level the expression of L chains may have been impaired. By LM-PCR (Fig. 4A), we first showed that inhibition of BCR-ABL1 kinase activity induces DNA double-strand breaks at RSS within the IGK locus after 8 h (Fig. 5A). For validation of the LM-PCR assay, PCR products were cloned and five randomly chosen clones were sequenced. Sequence analysis confirmed J1 RSS-linker ligation products (not shown). Such RSS-specific DNA strand breaks represent an initial step within the recombination process of IGK genes and demonstrate that the IGK locus is indeed targeted by the recombination machinery upon treatment with STI571. Because J-RSS-specific DNA strand breaks represent a precondition for V-J gene rearrangements, we next amplified V-J rearrangements from genomic DNA of B cell precursor leukemia cells treated with or without STI571 (Fig. 4B). Although sporadic V-J and V-J joints were already detectable under control conditions, V-J rearrangements at both IGK and IGL loci were clearly inducible by STI571 (Fig. 5B). As a specificity control, genomic DNA of BCR-ABL1+ chronic myeloid leukemia cells was also subjected to amplification of V-J and V-J rearrangements but no product was obtained. Integrity of DNA isolated from chronic myeloid leukemia cells was verified by amplification of a C1 germline fragment of the TCR locus (Fig. 5B). In agreement with a previous study on the effect of STI571 on v-abl-transformed murine pre-B cell lines (21), we observed a polyclonal pattern of V and V gene rearrangement upon STI571 treatment. STI571-induced V-J gene rearrangements involved all six IGK and all nine IGL V gene families. Whether IGK and IGL genes were also rearranged at similar frequencies in response to STI571 treatment remains unclear, because the PCR approach used here was not quantitative.
Rearrangement of the KDE may lead to inactivation of productively recombined V-J joints and hence represents another level at which Ig L chain expression may be impaired (15). To assess whether pre-existing functional V-J joints may have been inactivated by rearrangement of the KDE, we analyzed both types of KDE recombination within the IGK locus (Fig. 4C). The KDE can rearrange either to an RSS site within the intron region between the cluster of J gene segments and the intronic enhancer or to an RSS site immediately flanking a V gene segment (Fig. 4C). KDE rearrangement leads to deletion of both enhancers and the C gene in the former (J intron RSS-KDE) or to deletion of a V-J joint in the latter case (V RSS-KDE). To investigate whether KDE rearrangement may contribute to reduced L chain usage in STI571-treated leukemia cells, we amplified specific DNA fragments for KDE-germline configuration, J intron RSS-KDE rearrangement, and V RSS-KDE rearrangement from three BCR-ABL1+ leukemia cell lines (BV173, NALM1, SUP-B15) in the absence or presence of STI571 (Fig. 5C). Although KDE was found in germline configuration in untreated leukemia cells, STI571-mediated inhibition of BCR-ABL1 induced deletion of IGK alleles by rearrangement of the KDE to J intron RSS sites and to V RSS sites. Targeting of the IGK locus by the recombination machinery and generation of V-J joints appears to be normal, but overrepresentation of L chains can be attributed to extensive KDE rearrangement observed here (Fig. 5C). In the absence of BCR-ABL1 kinase activity, differentiating leukemia cells seem to undergo multiple rounds of rearrangement targeting both and L chain loci, which ultimately leads to preferential L chain expressiondue to KDE-mediated deletion of potentially productive V-J joints.
Rearrangements at the IGK and IGL loci occur in sequential order
For this reason, we considered STI571-induced Ig L chain gene rearrangement as a model for the pre-B to immature B cell transition in normal B cell development and investigated whether IGK, KDE, and IGL gene rearrangements follow a defined sequence of events. Sequential rearrangement of IGK, KDE, and IGL genes would be predicted by the ordered model of L chain gene recombination. To the contrary, according to the stochastic model of L chain gene rearrangement, IGK, KDE, and IGL genes would be recombined independently at each locus. Therefore, we incubated the three BCR-ABL1+ pre-B ALL cell lines in the presence or absence of STI571 and analyzed genomic DNA for RSS-specific double-strand breaks within the IGK, KDE, and IGL loci at different time points (Fig. 6). In the three cell lines, RSS-specific DNA double-strand breaks were already detectable at the IGK locus within 1 h after STI571 treatment. RSS breaks flanking the KDE followed after 6 h of STI571 treatment and RSS-DNA breaks were found at the IGL locus only after 12 h (Fig. 6). These findings are consistent with a sequential order of rearrangement events: ablation of BCR-ABL1 kinase activity almost immediately initiates IGK gene rearrangements, many of which are subsequently deleted by KDE- and ultimately replaced by IGL-gene rearrangements (Fig. 6). Of note, up-regulation of C germline transcripts did not precede the onset of KDE RSS breaks (Figs. 2A and 6). Unlike normal B cell development, only very few STI571-treated leukemia cells seem to stay at a + stage, while the vast majority of cells continue to rearrange, first KDE and ultimately IGL genes. It should be noted that between 12 and 24 h of STI571 incubation, RSS breaks could be detected concomitantly in IGK, KDE, and IGL loci (Fig. 6). Therefore, we cannot exclude that during this period of time, IGL gene rearrangements may have occurred before recombination within the IGK locus. Stability of RSS-strand break intermediates is cell cycle-dependent and mainly found in G1 and G0 phases (29). Indeed, STI571 treatment induces cell cycle arrest in BCR-ABL1+ B cell precursor leukemia cells (25). Thus, the detection of J-RSS breaks for an extended period of time (1–24 h), partially overlapping with KDE-RSS and even J-RSS breaks, can be explained by cell cycle arrest and increased stability of RSS breaks induced by STI571.
Indication for secondary IGK gene rearrangements
In contrast, the relatively wide time frame during which IGK gene rearrangements can occur might reflect that the leukemia cells are not all synchronized with respect to STI571-induced IGK gene rearrangement. This is consistent with a rather heterogenous pattern of RAG1 up-regulation following treatment with STI571 (Fig. 2). Moreover, RSS-specific DNA-strand breaks within the IGK locus do not necessarily reflect a single definitive V-J gene rearrangement and would also arise from any secondary recombination event during L chain revision (Fig. 4B). To determine whether ongoing L chain editing may extend the time window for RSS-specific DNA-strand breaks, we searched for traces of secondary rearrangements within the IGK locus. A pre-existing V-J joint can only be replaced by a rearrangement which uses an upstream-located V- together with a downstream-located J segment (Fig. 4B). Individual V segments are not arranged in the order of V gene families within the IGK locus. Therefore, we only compared the usage of upstream J1 and J2 segments to downstream J5 elements (see Fig. 4B) at different time points after STI571-induced initiation of L chain gene rearrangement. Consistent with a shift from upstream to downstream J elements, J1 and J2 gene rearrangements were detected slightly earlier than rearrangements involving the J5 gene segment (Fig. 7). Given that the two round PCR approach used here for analysis of V-J gene rearrangements is not quantitative, the relative amount of PCR products does not necessarily reflect the usage of individual J segments.
FIGURE 7. Shifting the preferential J usage from upstream to downstream located J gene segments indicates secondary IGK gene rearrangements. V1 gene rearrangements were amplified from three BCR-ABL1+ pre-B ALL cell lines using V1 leader- and V1 framework region I-specific primers together with either primers matching to J1 and J2 or primers matching to J5. As controls, PCRs were performed to which water was added instead of DNA.
Concluding remarks
We conclude that inhibition of BCR-ABL1 initiates a coordinated sequence of Ig L chain gene rearrangement events: almost immediately after ablation of BCR-ABL1 kinase activity by STI571, the IGK locus opens and is targeted by the recombination machinery. The continuous generation of new J RSS-specific DNA strand breaks even after 24 h together with a shift from upstream J1 and J2 elements to downstream J5 segments suggests that initial V-J rearrangements are followed by at least one, perhaps multiple, round(s) of secondary rearrangements. A possible initiation signal for subsequent KDE rearrangement could be exhaustion of the recombination potential of IGK alleles in multiple rounds of V-J rearrangement. Recombination of the KDE deletes V-J rearrangements from the chromosome and may initiate V-J gene rearrangements, which can first be detected after 12 h. According to these observations, it is not surprising that the vast majority of leukemia subclones that have been primed to differentiate, ultimately express L chains on their surface (Fig. 3). Obviously, the leukemia cells, unlike normal PBC, are lacking a feedback mechanism which prompts them to halt the recombination machinery in the presence of a productively rearranged IGK allele. Upon inhibition of BCR-ABL1, the leukemia cells seem to enter a pre-determined (ordered) sequence of rearrangement events, in which -expressing cells would only exist as a transition stage, inevitably leading to the expression of L chains.
Acknowledgments
We thank Stefanie Jauch and Peter Wurst for excellent technical assistance, Mario Fabri, Hassan Jumaa, Klaus Rajewsky, and Michael Reth for critical discussions, Martin Kr?nke and Janet D. Rowley for continuous support and discussions, and Sean McCleary for proofreading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Deutsche Forschungsgemeinschaft through Grants MU1616/2-1 and MU1616/3-1 (to M.M.), the German José-Carreras-Leukemia-Foundation (grant to M.M.), and the Ministry of Science and Research for North Rhine-Westphalia through the Stem Cell Network North-Rhine-Westphalia (to M.M.). F.K. is supported by scholarships of the Studienstiftung des deutschen Volkes and the K?ln Fortune program of the Faculty of Medicine of the University of Cologne. M.M. is supported by the Deutsche Forschungsgemeinschaft through the Emmy-Noether-Program.
2 Address correspondence and reprint requests to Dr. Markus Müschen, Laboratory for Molecular Stem Cell Biology, Building 14.80, Heinrich-Heine-Universit?t Düsseldorf, 40225 Düsseldorf, Germany. E-mail address: markus.mueschen@uni-duesseldorf.de
3 Abbreviations used in this paper: IGH, Ig μ H chain; PBC, pre-B cell; IGK, Ig L chain; IGL, Ig L chain; KDE, -deleting element; RSS, recombination signal sequence; ALL, acute lymphoblastic leukemia; SAGE, serial analysis of gene expression; HSC, hemopoietic progenitor cell; CMP, common myeloid progenitor cell; NBC, naive B cell; GCB, germinal center B cell; MBC, memory B cell; PC, plasma cell; TLP, T lymphoid progenitor; LM-PCR, ligation-mediated PCR.
Received for publication March 4, 2004. Accepted for publication October 18, 2004.
References
Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.
Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature 381:751.
Alt, F. W., G. D. Yancopoulos, T. K. Blackwell, C. Wood, E. Thomas, M. Boss, R. Coffman, N. Rosenberg, S. Tonegawa, D. Baltimore. 1984. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 3:1209.
Langman, R. E., M. Cohn. 1995. The proportion of B-cell subsets expressing and light chains changes following antigenic selection. Immunol. Today 16:141
Nadel, B., P. A. Cazenave, P. Sanchez. 1990. Murine gene rearrangements: the stochastic model prevails over the ordered model. EMBO J. 9:435.
Arakawa, H., T. Shimizu, S. Takeda. 1996. Re-evaluation of the probabilities for productive arrangements on the and loci. Int. Immunol. 8:91.
Gorman, J. R., N. van der Stoep, R. Monroe, M. Cogne, L. Davidson, F. W. Alt. 1996. The Ig enhancer influences the ratio of Ig versus Ig B lymphocytes. Immunity 5:241.
Inlay, M., F. W. Alt, D. Baltimore, Y. Xu. 2002. Essential roles of the light chain intronic enhancer and 3' enhancer in rearrangement and demethylation. Nat. Immunol. 3:463.
Zou, Y. R., S. Takeda, K. Rajewsky. 1993. Gene targeting in the Ig locus: efficient generation of chain-expressing B cells, independent of gene rearrangements in Ig . EMBO J. 12:811
Chen, J., M. Trounstine, C. Kurahara, F. Young, C. C. Kuo, Y. Xu, J. F. Loring, F. W. Alt, D. Huszar. 1993. B cell development in mice that lack one or both immunoglobulin light chain genes. EMBO J. 12:821.
Korsmeyer, S. J., P. A. Hieter, S. O. Sharrow, C. K. Goldman, P. Leder, T. A. Waldmann. 1982. Normal human B cells display ordered light chain gene rearrangements and deletions. J. Exp. Med. 156:975.
Korsmeyer, S. J., P. A. Hieter, J. V. Ravetch, D. G. Poplack, T. A. Waldmann, P. Leder. 1981. Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc. Natl. Acad. Sci. USA 78:7096.
Look, A.T.. 1997. Oncogenic transcription factors in the human acute leukemias. Science 278:1059.
Smith, K. M., R. Yacobi, R. A. Van Etten. 2003. Autoinhibition of Bcr-Abl through its SH3 domain. Mol. Cell. 12:27
LeBien, T. W.. 2000. Fates of human B-cell precursors. Blood 96:9.
Chen, Y. Y., L. C. Wang, M. S. Huang, N. Rosenberg. 1994. An active v-abl protein tyrosine kinase blocks immunoglobulin light-chain gene rearrangement. Genes Dev. 8:688.
Muljo, S. A., M. S. Schlissel. 2003. A small molecule Abl kinase inhibitor induces differentiation of Abelson virus-transformed pre-B cell lines. Nat. Immunol. 4:31.
Druker, B. J., C. L. Sawyers, H. Kantarjian, D. J. Resta, S. F. Reese, J. M. Ford, R. Capdeville, M. Talpaz. 2001. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:1038.
Hofmann, W. K., S. de Vos, D. Elashoff, H. Gschaidmeier, D. Hoelzer, H. P. Koeffler, O. G. Ottmann. 2002. Relation between resistance of Philadelphia-chromosome-positive acute lymphoblastic leukaemia to the tyrosine kinase inhibitor STI571 and gene-expression profiles: a gene-expression study. Lancet 359:481.
Klein, F., N. Feldhahn, L. Harder, H. Wang, M. Wartenberg, W. K. Hofmann, P. Wernet, R. Siebert, M. Müschen. 2004. The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells. J. Exp. Med. 199:673.
Deininger, M. W., S. A. Vieira, Y. Parada, L. Banerji, E. W. Lam, G. Peters, F. X. Mahon, T. Kohler, J. M. Goldman, J. V. Melo. 2001. Direct relation between BCR-ABL tyrosine kinase activity and cyclin D2 expression in lymphoblasts. Cancer Res. 61:8005
Müschen, M., S. Lee, G. Zhou, N. Feldhahn, V. S. Barath, J. Chen, C. Moers, M. Kr?nke, J. D. Rowley, S. M. Wang. 2002. Molecular portraits of B cell lineage commitment. Proc. Natl. Acad. Sci.(Florian Klein, Niklas Fel)