Functional screening identifies CRLF2 in precursor B-cell acute lymphoblastic leukemia

Results

Mutated CRLF2 Is a Gain-of-Function Oncoprotein in Poor-Prognosis B-ALL.

We identified CRLF2 in a functional screen for leukemia-derived cDNA that activate tyrosine kinase signaling (Fig. 1A(). In this screen, we infect the murine IL3-dependent cell line BaF3 with retroviral cDNA libraries constructed from bone marrow aspirates involved with more than 80% tumor. Clones that survive IL3 withdrawal invariably harbor tumor-derived cDNAs that obviate the requirement for IL3. After infection with a cDNA library constructed from a B-ALL specimen with 46, XY karyotype, we identified IL3-independent clones that contained a mutated, full-length cDNA transcript of CRLF2.

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Fig. 1.

Identification of CRLF2 in B-ALL. (A) Tumor-derived cDNA is packaged into retroviral particles that are used to infect BaF3 cells. Integrants (gray) survive in puromycin selection. Surviving clones (black) are isolated after IL3 withdrawal and their integrated cDNAs are sequenced and repackaged within retrovirus and confirmed. (B) IHC using anti-CRLF2 antibody and interphase FISH. ALL-73 has no detectable CRLF2 expression by IHC, whereas both ALL-74 and ALL-76 have visible expression and surface localization. (Left) FISH demonstrates 2 fusions of the CRLF2 (yellow) and IGH (green) probes in ALL-74, consistent with a reciprocal translocation, but not in ALL-73. (Right) FISH shows one probe centromeric to CRLF2 (yellow) lost in ALL-76, consistent with an intrachromosomal deletion.

We used a combination of quantitative (q) RT-PCR, immunohistochemistry (IHC), and gene expression profiling (GEP) to assay CRLF2 expression in adult B-ALL samples from Dana-Farber Cancer Institute (DFCI; n = 97) and Gruppo Malattie Ematologiche dell’Adulto (GIMEMA; n = 157) cohorts (Fig. 1B and Fig. S1). Cases with CRLF2 overexpression were clearly distinct, and overexpression correlated completely between assays (Fig. 1B and Fig. S1). Overall, CRLF2 was overexpressed in 15 of 120 (12.5%) adult B-ALL cases that lacked characteristic gene rearrangements, compared with 0 of 134 with these rearrangements (P < 10⁻⁴). CRLF2 overexpression was not present in 69 cases of T-cell ALL assayed by GEP (P = 0.001 vs. 15 of 120).

We analyzed 90 adult patients with B-ALL that lacked characteristic rearrangements who had available demographic and outcome information, pooled from the DFCI (n = 20) and GIMEMA (n = 70) cohorts (Fig. 2A). CRLF2-overexpressing and nonoverexpressing cohorts had similar median age, sex distribution, and white blood cell counts at presentation. However, disease-free survival (Fig. 2B) was significantly shorter among patients with CRLF2 overexpression (median, 17.8 mo vs. 37.8 mo; P < 0.03). There was also a trend for shorter overall survival among these patients (median, 25.5 mo vs. not reached; P = 0.17). Thus, CRLF2 overexpression is a marker of poor outcome among patients with B-ALL that lack characteristic rearrangements.

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Fig. 2.

Demographic and outcome analysis for patients with B-ALL that lack characteristic rearrangements, based on CRLF2 expression. (A) A comparison of 90 adult patients with B-ALL who lack characteristic cytogenetic rearrangements, pooled from GIMEMA (n = 70) and DFCI specimens (n = 20). All values are at the time of diagnosis. Median follow-up for the full cohort is 39.4 months. P values were derived using the Wilcoxon test for quantitative variables and a Fisher exact test for qualitative variables. ¹Values were available for only 85 of 90 patients. (B) Disease-free survival and overall survival from the time of diagnosis were estimated using the Kaplan-Meier product-limit method and compared in univariate analysis by the log-rank test.

CRLF2 Overexpression in Pediatric B-ALL and Other Lymphoid Malignancies.

To determine the frequency of CRLF2 overexpression in pediatric B-ALL, we reviewed the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) for B-ALL samples assayed on the Affymetrix U133 platform. Affymetrix HG-U133A and HG-U133Aplus2 arrays contain a single probe set (208303_s_at) that targets the CRLF2 transcript (both complete and partial coding sequence, as well as expression sequence tags).

We identified 9 datasets (Table S1 and Table S2) with a total of 1,253 pediatric ALL cases. Among the 3 datasets that included only high-risk patients, 52 (14.8%) of 351 B-ALL cases that lacked characteristic rearrangements had CRLF2 overexpression compared with 0 of the remaining 130 B-ALL cases (P < 10⁻⁴). This may underestimate the frequency of CRLF2 overexpression in the cohort without characteristic rearrangements, as some datasets did not distinguish patients based on karyotype, so patients with characteristic rearrangements were presumably included in the cohort of 351 B-ALL cases. In the six datasets that included both standard-risk and high-risk patients, CRLF2 overexpression was present in only 23 of 559 (4.1%) cases that lacked characteristic rearrangements (P < 10⁻⁴ vs. 52 of 351 from the high-risk–only datasets). In addition, 0 of 51 T-ALL cases overexpressed CRLF2 (P = 0.002 vs. 52 of 351 from the high-risk–only datasets).

We next asked whether CRLF2 is overexpressed in other lymphoid malignancies. We selected a cross-section of chronic lymphocytic leukemia (CLL) specimens, based on karyotype, IGHV somatic hypermutation, ZAP-70 and CD38 expression, CLL family history, and clinical features (Table S3). All 30 specimens had no detectable CRLF2 mRNA. A review of gene expression profiles from GEO dataset GSE6477 (n = 162) failed to identify significant CRLF2 expression in normal plasma cells, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma (MM), newly diagnosed MM, or relapsed MM. Low or undetectable CRLF2 expression was also confirmed in a panel of T-ALL (n = 22) and other (n = 14) cell lines (Table S4).

CRLF2 Locus Rearrangements.

Russell et al. (23) recently demonstrated that the CRLF2 locus, which is located in the pseudoautosomal regions of chromosomes X and Y, can undergo two types of rearrangement: intrachromosomal deletion or translocation with the Ig heavy chain (IGH) locus. Mullighan et al. (22) subsequently mapped the deletions and demonstrated that they juxtapose CRLF2 3′ of the first noncoding exon of P2RY8. In both cases, the chromosome X/Y breakpoints are upstream of CRLF2 and place the full CRLF2 coding sequence under alternate transcriptional control. CRLF2 and IGH are close to the telomeres of chromosome X/Y and 14, respectively. Thus, we designed a FISH strategy using probes against regions flanking CRLF2 and IGH (Fig. S2). Whereas the CRLF2 and IGH loci are clearly separate in cells that lack CRLF2 expression (Fig. 1B), FISH in 3 of 6 B-ALL specimens with high CRLF2 expression demonstrated joining of CRLF2 and IGH probes, consistent with a reciprocal chromosomal translocation (Fig. 1B). Two of the 3 remaining specimens had loss of a centromeric chromosome X/Y probe, consistent with an intrachromosomal deletion (Fig. 1B). The final specimen had neither a deletion nor a translocation, suggesting an additional mechanism for CRLF2 overexpression.

CRLF2/IGH translocations are akin to lymphoma-associated rearrangements involving BCL2 and BCL1 that result from aberrant V(D)J recombination (24). In 6 specimens, we PCR amplified der(14) translocation junctions between IGHJ segments on chromosome 14 and the region centromeric of CRLF2 on chromosome X/Y (Fig. 3A). Junctions involved sequence approximately 8 to 16 kb upstream of the CRLF2 translation start site, with multiple cases clustering near putative V(D)J recombinase recognition signal sequences (Fig. 3B). Thus, CRLF2/IGH translocations appear to result from aberrant V(D)J recombination that can involve cryptic recognition signal sequence in the pseudoautosomal regions.

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Fig. 3.

CRLF2/IGH junctions. (A) Junctions were amplified on der(14) using a common IgHJ primer and spaced primers 5′ (centromeric) of CRLF2 coding sequence. N nucleotides are nontemplated insertions. (B) Breakpoints for 4 of the 6 cases (black arrows) clustered near a single putative V(D)J recombinase recognition signal sequence (RSS, blue). The expected sites of cleavage by the V(D)J recombinase are in red. Cases MUTZ-5 and 7243 were reported by Russell et al. (23).

CRLF2 Phe232Cys Is a Gain-of-Function Mutation.

Sequencing of CRLF2 in 35 B-ALL specimens, including 14 overexpressing cases, identified multiple single nucleotide variants. We assayed the function of the 4 nonsynonymous variants (711T > G, 746G > A, 789A > G, 984C > T) by retroviral expression in BaF3 cells. Of these, only CRLF2 711T > G (Phe232Cys; Fig. 4A) conferred cytokine independence in murine BaF3 and human UT7 megakaryoblastic leukemia cells (Fig. 4B). The 711T > G mutation was present in 3 of 14 overexpressing cases (21.4%) and was a somatic mutation. Sequencing of cDNA from all three cases demonstrated expression of the mutant allele (Fig. 4A). The other three nonsynonymous variants were polymorphisms, as they were present in germline specimens and were recovered from healthy donor peripheral blood lymphocytes (PBLs).

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Fig. 4.

CRLF2 and JAK2 mutations in B-ALL. (A) The CRLF2 Phe232Cys mutation that results from 711T > G is present in genomic and cDNA, as shown for ALL-40. (B) UT7 cells and BaF3 cells that stably express CRLF2 and/or JAK2 alleles were grown in the absence of GM-CSF (UT7) or IL3 (BaF3). (Bottom Right) TSLP 1 ng/mL was added at d 0 as indicated. Growth is the number of viable cells relative to the number of cells initially seeded on d 0. Error bars represent 1 SD. (C) Unmodified BaF3 cells (-) and BaF3 cells that stably express WT CRLF2, CRLF2 Phe232Cys (FC), and/or IL7R were grown in the presence of IL3 except for the lane marked (-IL3). Protein lysates were separated by gel electrophoresis in the presence of reducing or nonreducing conditions and immunoblotted with an anti-CRLF2 antibody. (D) The 14 sequenced cases with CRLF2 overexpression fall into 3 categories based on CRLF2 and JAK2. Sequence traces for 3 different JAK2 alleles amplified from genomic DNA are shown.

The CRLF2 Phe232 residue is near the junction of the extracellular and transmembrane domains. Mutations that introduce cysteine residues in this region of other receptor tyrosine kinases, such as the erythropoietin receptor, can activate signal transduction through intermolecular disulfide-bonded dimers (25, 26). To confirm that CRLF2 Phe232Cys promotes constitutive dimerization, we performed immunoblots in BaF3 cells expressing WT CRLF2 or CRLF2 Phe232Cys under both reducing and nonreducing conditions (Fig. 4C). Under nonreducing conditions, the molecular weight of the CRLF2 Phe232Cys band, but not the WT band, was doubled, consistent with constitutive dimerization through the cysteine residues.

JAK2 Mutations Are Highly Associated with CRLF2 Overexpression.

The absence of gain-of-function CRLF2 mutations in most cases with CRLF2 overexpression raised the possibility that other factors within the same signaling cascade may harbor mutations. JAK mutations were recently reported in children with B-ALL (11, 13, 14). Thus, we sequenced for previously identified mutations in JAK1 and JAK2 and identified JAK2 Arg683Gly (n = 4), Arg683Ser (n = 1), and Arg683Thr (n = 1) substitutions (Fig. 4D) in 6 of 14 adult B-ALL cases that overexpress WT CRLF2. Of note, expression of CRLF2 Phe232Cys and JAK2 mutant alleles was mutually exclusive (Fig. 4D), suggesting that they function within the same pathway.

Mutant JAK proteins can transform growth factor–dependent cells only when expressed in combination with a type I cytokine receptor (11). The specific receptor that the JAK associates with, along with the particular JAK mutation, can affect the transformed phenotype (27). Thus, we asked whether overexpression of CRLF2 is essential for B-ALL associated with mutant JAK2. We linked gene expression (GEO GSE11877) and JAK mutation status from a cohort of 207 patients with high-risk pediatric B-ALL (Fig. S3A) (3, 14). CRLF2 expression among the 207 patients was clearly bimodal, with overexpression in 29 cases (14.0%; Fig. S3A). Twenty of the 207 patients also had mutations in JAK1 (n = 3), JAK2 (n = 16), or JAK3 (n = 1) (14). Strikingly, all 16 patients (100%) with JAK2 exon 16 mutations overexpressed CRLF2, as did 2 of the 3 patients with JAK1 mutations.

CRLF2 and JAK2 Contribute to Cytokine-Independent Growth.

The finding that all cases of B-ALL with JAK2 mutations overexpressed CRLF2 raised the possibility that JAK2 associates directly with CRLF2, either in the presence or the absence of IL7R. The stoichiometry of a WT CRLF2/IL7R complex is believed to be 1:1 (17). RT-PCR and gene expression profiling demonstrated that IL7R expression did not differ between the CRLF2 overexpressing and nonoverexpressing cases (Fig. S3B). This argues that mutated JAK2 signals with CRLF2 in the absence of IL7R. To test this possibility, we coexpressed WT and mutant versions of CRLF2 and JAK2 in the presence or absence of IL7R in BaF3 cells (Fig. 4B). Although WT CRLF2 or mutant JAK2 alone were insufficient to confer IL3 independence in BaF3 cells, the combination readily transformed the cells to IL3-independent growth in the absence of IL7R. Of note, the combination of WT CRLF2 with WT JAK2 provided a growth advantage versus JAK2 alone (Fig. 4B).

We next asked whether CRLF2 Phe232Cys renders cells hypersensitive to TSLP. Unlike in cells that express CRLF2/IL7R, the addition of TSLP had no effect on the growth of BaF3 cells expressing CRLF2 Phe232Cys (Fig. 4B). However, TSLP promoted the growth of cells that express CRLF2 Phe232Cys and IL7R (Fig. 4B), suggesting that CRLF2 Phe232Cys retains the ability to signal within a heterodimer. In contrast, TSLP did not promote the growth of cells that express CRLF2/mutant JAK2 (Fig. S3C).

Both CRLF2 Phe232Cys and CRLF2/mutant JAK2 promoted the phosphorylation of the JAK targets STAT5 and ERK (Fig. 5A). Phosphorylation of STAT5 by CRLF2 Phe232Cys was less robust than by CRLF2/mutant JAK2 but comparable to CRLF2/IL7R/TSLP. CRLF2 Phe232Cys also promoted the up-regulation of transcriptional targets downstream of JAKs (BCL-xL and PIM1) to a comparable or greater extent than WT CRLF2/mutant JAK2 (Fig. 5B).

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Fig. 5.

Signal activation and dependence on JAK signaling. (A) BaF3 cells expressing CRLF2, IL7R, and/or JAK2 proteins were starved of cytokine for 6 h, starved then stimulated with cytokine (TSLP 20 ng/mL or IL3 500 pg/mL) or grown in the presence of TSLP 1 ng/mL. Protein lysates were subjected to immunoblotting with antibodies against total and phosphorylated (P-) JAK2, STAT5, AKT, and ERK. (B) Quantitative RT-PCR of transcriptional targets in BaF3 cells that stably express CRLF2, IL7R, and/or JAK2 proteins grown in the absence of cytokine, starved of cytokine for 6 h, or stimulated with TSLP 20 ng/mL after 6 h cytokine starvation. *P < 0.05 versus cells that express CRLF2/IL7R grown in the presence of TSLP. ^P < 0.05 versus CRLF2/JAK2R683G and CRLF2/JAK2R683S. (C) BaF3 cells that stably express BCR/ABL, CRLF2, and/or JAK2 were grown in the absence of cytokines except where +IL3 indicates 500 pg/mL IL3. Data represents the ratio of viable cells exposed to JAK inhibitor-1 (Calbiochem) or k252a at varying concentrations for 72 h, compared with the same cell line exposed to vehicle. Error bars indicate 1 SD.

Whereas cells expressing WT CRLF2/mutant JAK2 had constitutively phosphorylated JAK2, cells expressing CRLF2 Phe232Cys and CRLF2/IL7R cells treated with TSLP had no detectable phospho-JAK2 (Fig. 5A). Yet, cells expressing either the CRLF2 Phe232Cys or CRLF2/mutant JAK2 were highly sensitive to a small-molecule JAK inhibitor (Fig. 5C), suggesting that CRLF2 signaling involves a JAK other than JAK2. Cells expressing CRLF2 Phe232Cys had little or no AKT phosphorylation, in contrast with cells expressing CRLF2/mutant JAK2 (Fig. 5A). However, cells expressing CRLF2/IL7R grown in the presence of TSLP also had minimal or no AKT phosphorylation despite the fact that stimulation of these cells with TSLP after a brief cytokine starvation clearly promoted AKT phosphorylation (Fig. 5A). Thus, the absence of AKT phosphorylation in CRLF2 F232C-expressing cells may reflect a limitation in the sensitivity of immunoblotting. As expected, TSLP had no effect on STAT5, ERK, AKT, or JAK2 phosphorylation in the absence of IL7R (Fig. 5A).

CRLF2 Transcriptional Signature in Adult and Pediatric B-ALL.

To characterize the dysregulated genes associated with CRLF2 overexpression, we performed a supervised analysis of gene expression profiles from the 22 B-ALL cases (8 CRLF2 overexpressing, 14 nonoverexpressing) that were validated by qRT-PCR (Fig. S1B). CRLF2 overexpression defined a 130–probe set (105-gene) “CRLF2 adult signature” (Fig. S4A and Table S5). Up-regulated genes in the CRLF2 adult signature included CD10, PKC iota, and the STAT5-induced negative regulator SOCS2, whereas genes showing decreased expression included the class I and class II HLAs. Higher SOCS2 expression among CRLF2 overexpressing cases was confirmed by qRT-PCR (123.1 fold higher than donor PBLs vs. 53.5 fold for CRLF2 nonoverexpressing cases; P < 0.05). Interestingly, several agents with activity against PKC family kinases had selective toxicity in BaF3-CRLF2 Phe232Cys cells, compared with BaF3 cells expressing WT CRLF2 (Fig. 5C and Fig. S5).

We applied a similar supervised approach to identify differentially expressed genes based on CRLF2 expression in the pediatric B-ALL GEO datasets GSE11877 and GSE12995, and compared these with the CRLF2 adult signature of 105 genes using Gene Set Enrichment Analysis (GSEA). Both pediatric signatures showed significant enrichment of the adult set (P < 0.001; false discovery rate (FDR) of 0; Fig. S4B).

To identify pathways up-regulated in CRLF2-overexpressing B-ALL, we applied GSEA to the CRLF2 differential expression signatures (from both GSE11877 and GSE12995) using gene sets from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (28). Four pathways, all involved in cytokine receptor signaling, were identified as significant (P < 0.01 and FDR < 0.10) in both datasets: “cytokine–cytokine receptor interaction,” “JAK-STAT signaling,” “neuroactive ligand receptor interaction,” and “ECM receptor interaction.” The enrichment of the JAK-STAT signaling pathway (Fig. S4C) further suggests that CRLF2 overexpression in B-ALL supports aberrant JAK-STAT activation. In agreement with the BaF3 data (Fig. 5B), BCL-xL and PIM1 were among the up-regulated genes enriched in the JAK-STAT signaling pathway.

Finally, we hypothesized that CRLF2 overexpression promotes a similar gene expression pattern to BCR/ABL. To address this, a BCR/ABL signature was obtained from the Oncomine Concepts Map (http://www.oncomine.org), which identified the top up-regulated and down-regulated genes (29). Using GSEA, the pediatric CRLF2 overexpression signatures from both datasets showed striking enrichment of the BCR/ABL-associated genes (P < 0.001, FDR = 0; Fig. S4D). Together, these findings establish common expression patterns between BCR/ABL–positive and CRLF2-overexpressing B-ALL in both children and adults.