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

Abstract

The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL-derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications.

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.

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.

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).

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.

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.