Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms - PubMed (original) (raw)

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

Stephen T Oh et al. Blood. 2010.

Abstract

Dysregulated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F-negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34(+) early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

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Figures

Figure 1

Figure 1

LNK mutations cause dysregulated TPO-dependent growth and JAK2-STAT3/5 activation. (A) Schematic of WT and the LNK DEL, E208Q, and R392E mutants is shown. WT LNK includes an N-terminal proline-rich dimerization domain (Pro/DD), a PH domain, an SH2 domain, and a C-terminal conserved tyrosine residue (Y). The DEL mutation leads to a frameshift and premature stop codon, resulting in loss of the PH and SH2 domains. The E208Q mutation localizes to the PH domain; R392E is a synthetic point mutation that disrupts the SH2 domain. In B through D, BaF3-MPL cells were transduced with EV, WT LNK, or mutant LNK (DEL, E208Q, R392E). Reproducible results were obtained for at least 3 independent experiments. (B) BaF3-MPL cells were washed in cytokine-free media and replated in the presence of concentrations of TPO ranging from 0 to 10 ng/mL. Cumulative growth of BaF3-MPL cells over 4 days is shown. (C) BaF3-MPL cells were starved overnight in cytokine-free media and stimulated with TPO (1 ng/mL) for the durations indicated, followed by measurement of JAK2, STAT3, and STAT5 activation via phospho-specific flow cytometry. Histograms for phosphorylated forms of JAK2 (pJAK2), STAT3 (pSTAT3), and STAT5 (pSTAT5) are displayed, with internal color representing fold-change in median fluorescence intensities (MFI) compared with unstimulated cells (Unstim) for each cell line. Red lines denote the MFI of EV unstimulated cells for comparison. (D) BaF3-MPL cells were cultured in the presence of TPO (10 ng/mL) and dimethyl sulfoxide (DMSO) or concentrations of JAK inhibitor I ranging from 0.2μM to 5μM. Cumulative growth at 4 days (normalized to maximal growth for each cell line) is shown. Error bars represent the SD of 2 replicates per sample in panels B and D.

Figure 2

Figure 2

Identification of a cytokine-responsive pSTAT3+/pSTAT5+ population in CD34+ early progenitors from patients with LNK mutations. Peripheral blood (PB) samples from patients with the LNK DEL and E208Q mutations, as well as PMF patients with the JAK2 V617F and MPL W515L mutations, were compared with normal donor. CD3−/CD66−/CD33mid immature myeloid cells are shown in panels A through C. (A) Samples were preincubated with DMSO or JAK inhibitor I (5 μM) for 30 minutes, and then stimulated with TPO (50 ng/mL) or G-CSF (20 ng/mL) for 15 minutes, before assessment of STAT3 and STAT5 activation by phospho-specific flow cytometry. (B) CD34 and CD38 surface staining for the cytokine-responsive pSTAT3+/5+ (“responsive”) cells from DEL, in comparison to the nonresponsive cells (includes all cells other than pSTAT3+/5+ cells) is displayed. Cytokine-responsive cells are shown in green (TPO) and red (G-CSF). In panels A and B, numbers depicted in each quadrant represent the percentage of total cells present in each quadrant gate. (C) The frequency of CD34+ cytokine-responsive cells was quantified and is displayed as fold-change versus normal donor. (D) PB cells from DEL were stimulated with G-CSF, and 6 subsets were sorted by fluorescence-activated cell sorting (FACS), as defined by the surface markers and phosphorylated STAT proteins shown in the table. Not applicable (n/a) denotes surface markers that were not used to delineate that specific subset. DNA was isolated from each subset, and allele-specific quantitative PCR for the DEL mutation was performed. Allele burden for each subset is displayed. Error bars represent the SD of 3 replicates. (E) Schematic showing role of LNK in regulation of JAK-STAT signaling, and model depicting hypothetical mechanisms for LNK dysfunction is shown. (Left) Cytokine/receptor binding (eg, TPO/MPL) results in JAK-STAT activation, which then leads to recruitment of a negative feedback pathway, in which LNK binds to MPL and JAK2, thereby inhibiting downstream STAT activation. (Right) LNK mutations affecting the PH domain (depicted as a yellow line) may lead to mislocalization of LNK in the cytoplasm, thereby disrupting the ability of LNK to inhibit JAK-STAT signaling. As the dimerization domain is retained, mutant LNK forms may also sequester WT LNK, potentially resulting in a dominant-negative effect.

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References

    1. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054–1061. - PubMed
    1. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144–1148. - PubMed
    1. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779–1790. - PubMed
    1. Levine R, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7(4):387–397. - PubMed
    1. Pardanani A, Levine R, Lasho T, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood. 2006;108(10):3472–3476. - PubMed

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