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Sep 6, 2011

Mutations and Mechanisms Involved in Childhood T-Cell Acute Lymphoblastic Leukemia Identified

Mutations and Mechanisms Involved in Childhood T-Cell Acute Lymphoblastic Leukemia Identified

Team says IL7R mutations in 9% of T-ALL lead to ligand-independent JAK-STAT signaling. [© Gernot Krautberger - Fotolia.com]

  • Some 9% of cases of childhood T-cell acute lymphoblastic leukemia (T-ALL) display gain of function mutations in exon 6 of the IL7R gene, scientists claim. The international researchers, led by a team at the Instituto de Medicina Molecular in Portugal, claim the mutations effectively lead to an extra, unpaired cysteine residue on part of the interleukin 7 (IL-7) receptor (IL-7R), which can link up with a cysteine on another mutant protein via disulphide bonding.

    As a result of homodimerization, downstream signaling is triggered in the absence of normal ligand binding to the receptor, leading to uncontrolled cell proliferation and, in experimental mice injected with cells carrying the mutant IL7R gene, the development of T-ALL-like cancer, report João T Barata, Ph.D., and colleagues.

    Describing their findings in Nature Genetics, the researchers say the work should pave the way to the development of new drugs that target IL-7R-mediated signaling in T-ALL. Their initial studies in cultured cells suggested that experimental drugs that block the JAK1-STAT signaling pathway triggered by dimerization of the mutant protein block the resulting cell proliferation and lead to cell death. The published paper is titled “Oncogenic IL7R gain-of-function mutations in childhood.”

    Signaling mediated by IL-7 and its receptor, IL-7R, is essential for normal T-cell development and homeostasis, and in humans IL7-R-inactivating mutations result in severe combined immunodeficiency, the authors write. Prior studies have also suggested that IL-7 and IL-7R may contribute to T-cell leukemia progression.

    IL7R encodes the IL-7Rα portion of the receptor complex for IL-7, which is transcriptionally upregulated by Notch1, one of the most commonly mutated genes in T-ALL, the authors continue. Research has also suggested that IL7R is involved in Notch-mediated leukemia cell maintenance, while the suggestion that IL-7R-mediated signaling is involved in T-cell leukemia is further supported by studies showing that 18% of adult, and 2% of pediatric, T-ALL cases have activating mutations in JAK1, which encodes a tyrosine kinase that directly binds to IL-7Rα.

    Based on accumulated observations to date, Dr. Barata’s team hypothesized that gain-of-function mutations in IL7R may be present in some T-ALL cases. They initially analyzed the coding region of IL7R in 68 pediatric diagnostic T-ALL samples treated at the Centro Infantil Boldrini in Brazil. Seven percent of these carried heterozygous mutations in IL7R that exclusively affected exon 6 and manifested as in-frame insertions or deletions-insertions in the juxtamembrane-transmembrane domain at the interface with the extracellular region. The mutations were all somatic, as they were detected at diagnosis but not in samples from the same patients in remission, the authors note.

    Subsequent examination of IL7R exon 6 in separate cohorts confirmed the results and showed the presence of heterozygous mutations in 12 of 133 cases. In most of these, mutations were located within the same hotspot. “In total, 17 of 201 (9%) T-ALL samples from three independent cohorts had IL7R exon 6 mutations,” the authors state. “This frequency was independently confirmed by a parallel study describing IL7R mutations in 10.5% of T-ALL cases.”

    Further analysis of samples from both IL7R-mutated and nonmutated diagnostic T-ALL samples showed that in the IL7R-mutated samples there was significant enrichment of a gene set that is activated on IL-7 stimulation in normal lymphocytes. However, IL7R mutations weren’t more frequently associated with concomitant mutations in other genes that have previously been associated with T-ALL, including JAK1, PTEN, or Notch1, and/or FBXW7, nor was there any association between IL7R mutations and either response to initial prednisone therapy or patient survival.

    Triggering of IL-7R by IL-7 involves recruitment of both the IL-7Rα and γc subunits of the IL-7R complex, and consequent activation of the tyrosine kinases JAK1 and JAK3, and thus downstream pathways, the authors state. They therefore tested whether T-ALL–associated IL7R mutations promoted either constitutive signaling or increased responsiveness to IL-7. When they compared downstream effects in a IL7R-mutated T-ALL sample with those harboring wild-type IL7R, they found that the sample with the IL7R mutation showed constitutive JAK1 and STAT5 phosphorylation.

    Indeed, further studies in experimental cell lines confirmed that IL7R mutations are gain of function, inducing constitutive hyperactivation of IL-7R–mediated signal transduction independent of γc. While knockdown of JAK1 abrogated mutant IL7R-dependent constitutive STAT5 phosphorylation, JAK3, JAK2, and TYK2 were not activated by the IL7R mutants, indicating “that JAK1 is the only Janus kinase that is mandatory for signaling triggered by mutant IL-7Rα,” the authors suggest.

    At the protein level, 82% of the IL7R mutations identified resulted in an unpaired cysteine residue in the extracellular juxtamembrane-transmembrane interface region. In other receptors, mutations that introduce cysteines in the equivalent region have been implicated in intermolecular disulfide bond formation, resulting in homodimerization and signaling activation. Cell line assays with the IL-7Rα mutants suggested that these were also present mostly as dimers, and reducing the mutants to monomeric form led to downregulation of STAT5  phosphorylation. Dimerization and constitutive signaling could in addition be blocked by substituting the mutated cysteine with an alanine or serine.

    The researchers moved on to investigate the consequences of mutated IL7R and constitutive signaling at the cellular level. They found that expression of mutant, but not wild-type, IL-7Rα into IL-7–dependent cells and IL-3–dependent cells promoted both cell cycle progression and viability, and in the IL-3-dependent Ba/F3 cells conferred growth factor independence, “indicating that the IL7R mutants have a transforming capacity,” they write. Importantly, substituting the extra cysteine residue with a serine or alanine reversed the transforming capacity of the IL-7Rα mutants, “suggesting that intermolecular disulfide-bond–dependent homodimerization is mandatory not only for signaling but also for the functional effects of IL-7Rα mutants.”

    Critically, subcutaneous injection of mutant IL-7Rα–expressing cells into experimental mice resulted in tumor formation, with all cancer-bearing animals demonstrating a phenotype typical of T-ALL. The tumors were also transplantable into secondary recipient animals, and were not dependent on the presence of IL-7: injection of mutant IL-7Rα–expressing cells led to tumor development in IL-7–deficient mice. “Taken together, our results indicate that IL7R mutational activation is an oncogenic event involved in T-ALL,” the authors state.

    The team finally investigated the therapeutic applications of their findings by testing a number of JAK inhibitors on IL7R-mutant cell lines. Compounds tested included Pyridone 6 (JAK inhibitor I), and the clinical-stage candidates CP-690550 and INCB018424, which are currently being evaluated in clinical trials against rheumatoid arthritis and several cancers. All three drugs significantly downregulated JAK1 phosphorylation and consequent downstream activation of STAT5 and Akt, and induced cell death in a dose- and time-dependent manner.

    CP-690550, INCB018424, and the JAK inhibitor CYT387 also inhibited the proliferation of mutant IL-7Rα–expressing D1 cells, while the STAT5-specific small molecule inhibitor triggered the death of Ba/F3 cells expressing mutant IL-7Rα. Importantly, primary T-ALL cells with IL7R mutations were also sensitive to JAK-STAT pathway inhibition and, with the exception of CP-690550, all the drugs had significant cytotoxic effects on diagnostic leukemia cells.

    The authors maintain their studies represent “the first example of an oncogene in the γc family of cytokine receptors, which is critically involved in numerous lymphoid cell functions…our findings provide a strong rationale for specific targeting of IL-7R–mediated signaling as a treatment option for T-ALL.”


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