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-offunction 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.uring the past decade, studies using oligonucleotide arrays and high-throughput sequencing have identified several genetic and transcriptional aberrations in B-cell acute lymphoblastic leukemia (B-ALL) (1), leading to three conceptual advances. First, genes involved in normal B-cell development (e.g., PAX5, IKZF1) are frequently mutated in B-ALL (1-3). Second, B-ALL is highly heterogeneous and can exist as multiple, genetically distinct clones within the same individual (1, 4). Third, B-ALL transcriptional profiles cluster based on characteristic chromosomal rearrangements, hereafter defined as rearrangements of TEL, MLL, TCF3, and BCR/ABL (5-8).However, one third of B-ALL cases lack characteristic rearrangements (9). Transcriptional profiles from a subset of these leukemias cluster with profiles from BCR/ABL-expressing B-ALL (3, 5), suggesting that the former harbor cryptic alterations in tyrosine kinase signaling. Supporting this notion, mutations in JAKs were recently identified in a small percentage of pediatric B-ALL and approximately 20% of ALL in children with Down syndrome (10-14).Upon ligand binding to a type I cytokine receptor, JAKs phosphorylate substrates including STATs, which in turn affect the transcription of progrowth and antiapoptotic factors (15). JAK enzymatic activity requires interaction with a cytokine receptor, which is believed to se...

Newly diagnosed acute lymphoblastic leukemia in China (I): abnormal genetic patterns in 1346 childhood and adult cases and their comparison with the reports from Western countries

“…44 In contrast, in adult patients, the most frequent ones in B-ALL are t(9;22)/BCR --ABL that activates the tyrosine kinase signaling pathway, Ik6 variant of IKZF1 gene, and CRLF2 overexpression that is a cytokine receptor activating the JAK/STAT pathway. 14,15 Interestingly, CRLF2 overexpression and IKZF1 splicing deletion, which are significantly more involved in adults than in children, are both related to the poor outcome in B-ALL and closely associated with JAK mutations. 16,18,45 Analyzed by gene expression profiling, the three abnormal transcriptional signatures of CRLF2 overexpression, IKZF1 aberration and BCR --ABL demonstrated a high degree of similarity, suggesting the presence of similar pathogenic mechanisms with interference of the transcriptional regulations or signaling pathways that control the proliferation, survival and self-renewal of hematopoietic stem cells.…”

“…Each sample was tested in triplicate. The cut-off value of CRLF2 overexpression was identified according to Yoda et al 15 Genomic rearrangement was confirmed by fluorescence in situ hybridization for the detection of breakpoints of IGH@ locus (LSI IGH@ Dual Color Break-Apart Rearrangement Probe, Abbott Diagnostics, Chicago, IL, USA) and involvement of CRLF2 on chromosome X, 16 and genomic PCR for the PAR1 deletion resulting in fusion of P2RY8 --CRLF2 transcript, which was also confirmed by RT-PCR. 17,18 (Supplementary Figure 2).…”

Genomic profiling of high-risk acute lymphoblastic leukemia

“…P2RY8 is a member of a family of purinergic receptor genes that is expressed in hematopoietic cells, including leukemic blasts, and has previously been identified as a rare target of translocation to SOX5 in lymphoma. 72 CRLF2 alterations in B-progenitor ALL have been subsequently confirmed and identified by multiple groups, including adult ALL 61,70,71,73,74 (Table 1). CRLF2 is rearranged in five to seven percent of B-progenitor childhood ALL cases, most commonly by IGH@-CRLF2 rearrangement or the PAR1 deletion resulting in expression of P2RY8-CRLF2.…”

“…Less commonly, CRLF2 is rearranged to other, as yet unknown partner genes or harbors presumed activating mutations, most commonly F232C. 73,75 A striking observation is that CRLF2 alteration, most commonly the PAR1 deletion, is present in over 50% of ALL associated with Down's syndrome (DS-ALL), 61,74 in which other chromosomal rearrangements characteristic of childhood ALL are uncommon. 76 The basis for this increased frequency in DS-ALL is currently unknown.…”

“…61,71,73,74 Over half of CRLF2-rearranged cases harbor activating JAK1 or JAK2 mutations, and conversely, nearly all JAK-mutated cases have CRLF2 rearrangements, suggesting that these lesions together contribute to leukemogenesis. Importantly, in non-DS-ALL, CRLF2 alteration and JAK mutations are associated with the presence of IKZF1 alterations, and several studies have observed strong associations between CRLF2/JAK alterations and very poor outcome, 70,77 suggesting that JAK inhibition may be a useful therapeutic approach in these highrisk cases that at present frequently fail maximal therapy.…”