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Aneuploidy and translocations are hallmarks of B-progenitor acute lymphoblastic leukemia (ALL), but many patients lack a recurring chromosomal alteration. Here we report a recurring interstitial deletion of the pseudoautosomal region 1 of chromosomes X and Y in B-progenitor ALL that juxtaposes the first, non-coding exon of P2RY8 to the coding region of CRLF2 (which encodes cytokine receptor like factor 2, or thymic stromal lymphopoietin receptor). The P2RY8-CRLF2 fusion was identified in 7% of B-progenitor ALL cases, and was identified in over 50% of ALL cases arising in patients with Down syndrome (53% of 75 cases). CRLF2 alteration was associated with the presence of activating JAK mutations, and expression of P2RY8-CRLF2 together with JAK2 mutants resulted in constitutive Jak-Stat activation and cytokine-independent growth of Ba/F3-IL7R cells, indicating that these two genetic lesions together contribute to leukemogenesis in B-progenitor ALL.
Chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL), the commonest malignancy of childhood, and include aneuploidy (hyperdiploidy and hypodiploidy) and recurring chromosomal translocations, such as t(12;21) [ETV6-RUNX1], t(1;19) [TCF3-PBX1], t(9;22) [BCR-ABL1] and rearrangement of MLL1. These alterations are important events in leukemogenesis and influence response to therapy. However, up to one-quarter of childhood ALL cases lack a recurring chromosomal alteration, and the genetic basis of these cases is poorly understood.
To identify submicroscopic genetic alterations contributing to the pathogenesis of ALL, we previously performed high resolution profiling of DNA copy number alterations and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, and identified multiple recurring genetic alterations targeting key cellular pathways including lymphoid development, cell cycle regulation and tumor suppression2,3. These alterations included a novel deletion involving the pseudoautosomal region 1 (PAR1) of Xp22.3/Yp11.3 in 15 B-progenitor ALL cases lacking common chromosomal translocations. Notably, six of eight ALL cases associated with Down syndrome (DS-ALL) harbored this deletion.
To further characterize the PAR1 deletion, we expanded our analysis of DNA copy number alterations and LOH to include 329 ALL cases, including 272 B-progenitor (22 DS-ALL) and 57 T-lineage childhood ALL cases (see Supplementary Table 1 online for patient characteristics and Supplementary Table 2 online for a full listing of all DNA copy number alterations). The PAR1 deletion was identified in 19 (7%) B-progenitor ALL cases, including 12 (54.5%) of the 22 DS-ALL cases, all of which lacked recurring translocations commonly associated with non-DS ALL (Supplementary Table 3 online). Notably, all 10 DS-ALL cases with cytogenetic abnormalities commonly observed in DS-ALL4–6 (+X in 8 cases, and del(9)(p22) in 2 cases) had a PAR1 deletion.
The region of PAR1 deletion appeared identical in all cases (Fig. 1a), involved at least three genes (P2RY8, SLC25A6 and IL3RA, Fig. 1b), and was confirmed by genomic quantitative PCR (Supplementary Table 4 online). The deletion was adjacent to the cytokine receptor genes CSF2RA and CRLF2, but due to sparse probe coverage on the SNP array platform used (Fig. 1b), it was not possible to precisely define the extent of deletion using SNP array data alone.
To map the boundaries of deletion, we performed array-based comparative genomic hybridization on two samples using a one million feature oligonucleotide array with dense coverage of the region. This showed that the PAR1 deletion extended from immediately centromeric (upstream) of CRLF2 exon 1 to intron 1 of P2RY8 (Fig. 1b). CRLF2 encodes cytokine receptor like factor 2 (also known as thymic stromal lymphopoietin receptor, TSLPR), a lymphoid signaling receptor molecule that forms a heterodimeric complex with interleukin 7 receptor alpha (IL7RA) and binds thymic stromal lymphopoietin (TSLP)7,8. P2RY8 encodes a purinergic receptor (P2Y, G-protein coupled, 8) that is expressed at high levels in many tissues, including leukemic cells9. A single case of rearrangement of P2RY8 to SOX5 has been reported in primary splenic follicular lymphoma10. These observations suggested that the PAR1 deletion may result in a novel rearrangement involving P2RY8 and CRLF2.
RT-PCR confirmed the presence of chimeric transcripts juxtaposing the first, non-coding exon of P2RY8 to the entire coding region of CRLF2 in all cases with PAR1 deletion that had available material for analysis, but none of 50 cases that lacked the deletion (Fig. 1c, Supplementary Fig. 1 online). The fusion junction was identical in each case (Fig. 1d). Quantitative RT-PCR analysis demonstrated increased P2RY8-CRLF2 expression in cases with the PAR1 deletion (Supplementary Fig. 2 online). Moreover, elevated cell surface expression of CRLF2 was also detected by flow cytometric analysis of cryopreserved leukemic cells (Supplementary Fig. 3 online). A single DS-ALL case (DS-ALL-#14) was found to have elevated CRLF2 mRNA levels by real time PCR but lacked the deletion, suggesting an alternative mechanism of CRLF2 dysregulation. Rearrangements of the immunoglobulin heavy chain locus (IGH@) at 14q32.33 to CRLF2 have recently been reported in B-progenitor ALL11, and fluorescence in situ hybridization (FISH) analyses of interphase nuclei confirmed IGH@-CRLF2 rearrangement in this case (Supplementary Fig. 4 online). Thus, 13 (60%) of DS-ALL cases had genomic rearrangements resulting in dysregulated CRLF2 expression.
We next characterized the genomic breakpoints of the PAR1 deletion by long template PCR of genomic DNA extracted from leukemic cells (Supplementary Figs. 5 and 6 online). The deletion breakpoints were highly conserved, and were located 3.4kb upstream of CRLF2 exon 1, and 0.3–1kb distal to P2RY8 exon 1 (Supplementary Fig. 5c online). Notably, we observed partly or fully conserved heptamer recombination signal sequences (RSSs) immediately internal to the deletion breakpoints, and a variable number of non-consensus nucleotides between the aligning sequences in each case (Supplementary Figs. 5c and 6 online). These data suggest that the PAR1 deletion may arise as a result of aberrant activity of the antigen receptor recombinases encoded by the RAG genes (recombinase activating genes), a mechanism that has been implicated in the generation of other sentinel chromosomal rearrangements and deletions in ALL3,12.
DS-ALL is characterized by the presence of activating JAK mutations in up to 28% of cases, most commonly in the JAK2 pseudokinase domain at or around R68313–16. JAK1, JAK2 and JAK3 mutations have also been identified in 10% of high-risk B-progenitor ALL patients without Down syndrome16. In this cohort, we identified previously described and novel JAK mutations in 11 (4%) B-progenitor ALL cases, including six (27.2%) DS-ALL cases (Supplementary Fig. 7 online). These included mutations in the JAK2 pseudokinase domain (R683G, N=5, R683S, N=2 and IR682RG, N=1), JAK2 kinase domain (T875N and G861W, 1 each) and JAK1 pseudokinase domain (V658F, the homolog of JAK2 V617F). Moreover, we observed significant association between CRLF2 alterations and JAK mutations. Nine (45%) patients with CRLF2 alterations had JAK mutations, compared to two (0.6%) patients lacking CRLF2 lesions (Fisher exact P<0.0001; Table 1, Supplementary Table 1 online). Although we previously identified an association between outcome and the presence of JAK mutations in high risk pediatric ALL16, no association was observed between CRLF2 and/or JAK mutation status and outcome in DS-ALL (data not shown).
To validate the high frequency of CRFL2 and JAK mutations in DS-ALL, we examined a second cohort of 53 B-progenitor DS-ALL cases (Supplementary Table 5 online). Twenty-eight (52.3%) had the PAR1 deletion detected by genomic PCR (Supplementary Fig. 8a online). In cases with available material, the deletion was associated with elevated CRLF2 expression on microarray analysis (Supplementary Fig. 9 online) and expression of P2RY8-CRLF2 (Supplementary Fig. 8b online). Moreover, eight (27.6%) patients with the PAR1 deletion had JAK2 mutations, compared to one patient (4.2%) without the PAR1 deletion (Fisher exact P=0.02), again highlighting the remarkable association between CRFL2 overexpression and activating JAK2 mutations.
The co-occurrence of CRLF2 alteration and JAK mutations suggested that these events likely cooperate in leukemogenesis. To examine this hypothesis, we assessed the effect of P2RY8-CRLF2 and JAK mutations on the ability of the cytokine-dependent murine B-progenitor Ba/F3 cell line to grow in the absence of exogenous cytokine. JAK mutations are usually alone insufficient to transform Ba/F3 cells, but render the cells growth factor-indepedent when coexpressed with erythropoietin or thrombopoietin receptors13,15,16. As CRLF2 normally forms a heterodimeric complex with the interleukin 7 receptor alpha chain (IL7RA), we expressed IL-7 receptor alpha in Ba/F3 cells (Ba/F3-IL7R cells). Expression of P2RY8-CRLF2 or JAK2 mutants alone (either JAK2 R683G or the kinase domain mutation P933R16) in the Ba/F3-IL7R cells failed to induce cytokine-independent growth. In contrast, co-expression of both P2RY8-CRLF2 and JAK mutants resulted in constitutive Jak-Stat activation and cytokine-independent growth in Ba/F3-IL7R cells (Fig. 2, Supplementary Figs. 10 and 11 online). Moreover, this transformation was attenuated by pharmacological Jak inhibition (Fig. 2c) and knockdown of CRLF2 expression by short hairpin RNAs (Fig. 2d, Supplementary Fig. 12 online).
Together, these data describe a recurrent intra-chromosomal deletion of the pseudoautosomal region 1 of Xp22.3/Yp11.3 that results in the generation of a chimeric P2RY8-CRLF2 mRNA, and markedly elevated expression of CRLF2. CRLF2 overexpression from IGH@-CRLF2 rearrangement or PAR1 deletion has also been reported in B-progenitor ALL11. However, while CRLF2 alteration is uncommon in B-progenitor ALL (5% of cases), it is present in over 55% of cases of DS-ALL, in which chromosomal rearrangements characteristic of non-DS-ALL are uncommon4. Moreover, CRLF2 alteration was observed exclusively in cases lacking translocations associated with ALL, suggesting that CRLF2 alteration is a potent leukemogenic event in the setting of trisomy 21, and may account in part for the up to 20-fold increased risk of developing ALL in children with DS6,17. The associations of CRLF2 alteration with JAK mutations and additional recurring cytogenetic alterations in DS-ALL (gain of chromosome X, and del(9p))4 are also notable, and suggest that these lesions cooperate in leukemogenesis. Interestingly, three of five cases with PAR1 deletion and gain of chromosome X had evidence of two copies of the PAR1 deletion on FISH analysis (Supplementary Fig. 4e–g online), suggesting that gain of X results in duplication and high level expression of the P2RY8-CRLF2 fusion. Amplification of regions of chromosomal rearrangement resulting in gene fusion has previously been noted in acute leukemia, for example the NUP214-ABL1 translocation in T-lineage ALL18.
CRLF2 interacts with IL-7 receptor alpha to form a heterodimeric receptor for the cytokine thymic stromal lymphopoietin (TSLP). TSLP/CRLF2 signaling has important roles in T and dendritic cell development, inflammation and allergic disease7,19, and promotes B lymphoid proliferation, but may not be required for normal B cell development20,21. CRLF2 signaling results in downstream STAT5 phosphorylation20,22, and for the human receptor, phosphorylation of JAK220. An important observation is that in contrast to myeloproliferative diseases, in which homozygous mutation of JAK2 V617 is common23, the JAK mutations in B-progenitor ALL are usually heterozygous, and do not occur at JAK2 V61713,15,16. The basis of the disease specificity of the different JAK mutations is unknown, and it has been suggested that the different JAK2 pseudokinase domain mutations may facilitate interaction with different substrates and signaling pathways13. Detailed analysis of the transforming effect of mutant Jak alleles in Ba/F3 cells expressing P2RY8-CRLF2 and/or IL-7R indicated that most Jak mutations are not transforming in the absence of CRLF2 overexpression (the exceptions being Jak2 V617F and the homologous mutation Jak1 V658F; Supplementary Note and Supplementary Fig. 11 online). In contrast, the mutations most commonly observed in B-progenitor ALL - those at or near JAK2 R683 - require coexpression of P2RY8-CRLF2 for transformation in this assay. Notably, transformation (as measured by the rate of cytokine-independent cell growth) was more marked with concomitant IL7RA expression (Supplementary Fig. 11 online). Moreover, co-immunoprecipitation experiments using 3xFLAG-tagged P2RY8-CRLF2 demonstrated direct interaction of CRLF2 and phosphorylated Jak2 (Supplementary Fig. 13 online). Thus, our results indicate that aberrant signaling through the CRLF2–IL7R receptor, mediated by CRLF2 overexpression and Jak mutation, is a key event in B lymphoid transformation. Consistent with this, in a recent study identifying CRLF2 rearrangement by FISH in B-progenitor ALL, overexpression of CRLF2 promoted transformation and Stat5 phosphorylation of primary murine lymphoid progenitors11. This study examined JAK2 exon 14 mutational status in a small cohort of DS-ALL cases (N=24) and also observed an association between CRLF2 rearrangement and JAK mutations11, further supporting cooperativity of these lesions in leukemogenesis.
As a substantial proportion of cases with CRLF2 overexpression lack JAK mutations, mutational analysis of other kinases and mediators of JAK-STAT signaling is warranted. These findings also suggest that detection of increased CRLF2 expression will be a useful diagnostic strategy in ALL, and that JAK-STAT inhibition may be useful in the treatment of patients with B-progenitor ALL with CRLF2 and JAK mutations.
We thank Michael Wang and Deanna Naeve, Functional Genomics Laboratory, Hartwell Center, St Jude Children’s Research Hospital for performing array-CGH analysis; Emily Walker and John Morris, CACT Laboratory, Hartwell Center, St Jude, for performing SNP microarrays; Stephanie Tate, Jianling Armstrong and Karen Rakestraw of the St Jude Hartwell Center Sequencing Core for performing sequencing; the St Jude Flow Cytometry Core; John Gray and the St Jude Vector Core for lentiviral reagents and methods; the St Jude Tissue Resources Laboratory for providing primary patient samples; Stephen Nutt for providing the MSCV-mIL7R-IRES-hCD4 retroviral vector; and Malcolm Smith and Kevin Dobbin for gene expression studies of CRLF2. This work was supported by an NCI Cancer Center Support Grant (CCSG) P30 CA021765 and ALSAC of St Jude Children’s Research Hospital, a Bear Necessities Pediatric Research Foundation Grant (to K.R.R.), a Children’s Cancer Research Foundation Grant (to K.R.R.) and NIH Pediatric Oncology Clinical Research Training Grant CA90433-06 (to K.R.R.).
Aglient array-CGH data has been deposited in the NCBI’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), accession GSE16724. The P2RY8-CRLF2 mRNA sequence has been deposited in Genbank, accession GQ280263. SNP array data is available from the authors upon request.
AUTHOR CONTRIBUTIONSCGM designed and coordinated the study, designed assays, performed experiments, analyzed data and wrote the manuscript
JRC-U generated retroviral vectors and performed Ba/F3 assays
LAAP performed JAK sequencing and quantitative PCR assays
MLL performed PAR1 deletion genomic PCR
WL performed statistical analysis
JZ analyzed sequencing data
Jing Ma analyzed microarray data
EC-S performed flow cytometry and analyzed data
RCH and CLW developed FISH assays
Julia Meyer performed experiments and analyzed data
FMM, AJC and NAH performed FISH assays and analyzed cytogenetic data
RTW provided luciferase vectors
JC designed subcloning vectors
GB and AP provided patient samples
SCR performed cytogenetic analysis
SPH coordinated studies and sample collection
JRD provided patient samples
WLC provided patient samples, performed experiments and analyzed data
KRR provided samples, performed experiments and analyzed data