Gain-of-function mutations in interleukin-7 receptor-α (IL7R) in childhood acute lymphoblastic leukemias

Abstract

Interleukin-7 receptor α (IL7R) is required for normal lymphoid development. Loss-of-function mutations in this gene cause autosomal recessive severe combined immune deficiency. Here, we describe somatic gain-of-function mutations in IL7R in pediatric B and T acute lymphoblastic leukemias. The mutations cause either a serine-to-cysteine substitution at amino acid 185 in the extracellular domain (4 patients) or in-frame insertions and deletions in the transmembrane domain (35 patients). In B cell precursor leukemias, the mutations were associated with the aberrant expression of cytokine receptor-like factor 2 (CRLF2), and the mutant IL-7R proteins formed a functional receptor with CRLF2 for thymic stromal lymphopoietin (TSLP). Biochemical and functional assays reveal that these IL7R mutations are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells. A cysteine, included in all but three of the mutated IL-7R alleles, is essential for the constitutive activation of the receptor. This is the first demonstration of gain-of-function mutations of IL7R. Our current and recent observations of mutations in IL7R and CRLF2, respectively suggest that the addition of cysteine to the juxtamembranous domains is a general mechanism for mutational activation of type I cytokine receptors in leukemia.

Figure 1.

Somatic mutations of IL7R (NM_002185.2) in patients with BCP-ALL. (A) IL7R mutation localization. Amino acid numbers of the different domains are indicated. (B) Sequences of IL7R insertions and deletions mutations at the transmembrane domain. The inserted sequences are within brackets (All Ins-Del mutations were heterozygous, only the mutated allele is shown after allele separation). N, sites with both WT and mutant allele at the same position; Ins, insertion; Del, deletion. (C) Alignment of WT and mutated IL7R transmembrane domain sequences. Numbers show the positions of nucleotides and corresponding amino acids. The inserted amino acids are shown in red, with the cysteine residue in bold. (D) Expression of mutations: Examples of two mutated IL-7R sequences with S185C and c.814 Ins 13 Del A. The mutated allele is expressed in the RNA from diagnosis, but not in remission samples.

Figure 2.

IL7R mutations are gain-of-function mutations. (A) FACS analysis of BaF3 cells stably transduced with CRLF2 and either IL-7R WT, IL-7R S185C, or IL-7R InsPPCL in the presence of IL-3. The same cells were then grown without IL-3 for 1 wk and then analyzed again. (B) Cytokine withdrawal assay of BaF3 and BaF3-CRLF2 cells transduced with either IL-7R WT, IL-7R S185C, or IL-7R InsPPCL. Error bars represent SE. (C) Constitutive phosphorylation of Stat5 and ribosomal protein S6 (RPS6) in BaF3 and BaF3-CRLF2 cells expressing IL-7R mutants, after 5 h of cytokine deprivation. IL-3⁺ indicates cells harvested after 5 h of IL-3 deprivation followed by 20 min of IL-3 stimulation. All experiments were repeated four times.

Figure 3.

CRLF2 and mutated IL7R form a functional TSLP receptor and sensitize cells to TSLP. (A) BaF3 cells expressing CRLF2 and either WT or mutated IL-7R were starved of IL-3 for 5 h, and then treated or not with 100 ng/ml TSLP where indicated for 25 min. (B) BaF3-CRLF2 cells transduced with either WT IL-7R or IL-7R S185C were treated with increasing doses of TSLP in the absence of IL-3. The number of cells was normalized to day 0 (therefore relative growth was measured). Experiments were performed three times. Error bars indicate SE.

Figure 4.

Functional significance of the cysteine residue in mutated IL7R. (A) FACS analysis of BaF3-CRLF2 cells expressing either S185G or IL-7R InsPPGL. In these constructs, the cysteine was mutated to glycine. (B) Growth assay of BaF3 and BaF3-CRLF2 cells transduced with IL-7R WT, IL-7R S185G, or IL-7R InsPPGL in the presence or absence of 30 ng/ml TSLP and the absence of IL-3. Error bars indicate SE. (C) BaF3 and BaF3-CRLF2 cells expressing indicated IL-7R mutants were incubated without IL-3 for 5 h. Where indicated, 100 ng/ml TSLP was added for 25 min. (D) Homodimerization of IL-7R InsPPCL mutant under nonreducing conditions. BaF3 cells that stably express CRLF2 with IL-7R (WT or mutated), IL-7R InsPPCL alone, or CRLF2 F232C (that has been shown previously to homodimerize; Yoda et al., 2010), were grown in the presence of IL-3. Protein lysates were separated by gel electrophoresis in the presence of nonreducing conditions and immunoblotted with an anti-CRLF2 or anti–IL-7R antibody. All experiments were performed three times.