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. 2016 May;30(5):1204-8.
doi: 10.1038/leu.2015.263. Epub 2015 Sep 30.

Activating somatic mutations outside the SH2-domain of STAT3 in LGL leukemia

E Andersson  1   2 , H Kuusanmäki  1   3 , S Bortoluzzi  1 , S Lagström  3 , A Parsons  3 , H Rajala  1   2 , A van Adrichem  3 , S Eldfors  3 , T Olson  4 , M J Clemente  5 , A Laasonen  2 , P Ellonen  3 , C Heckman  3 , T P Loughran  4 , J P Maciejewski  5 , S Mustjoki  1   2   6
Affiliations

Activating somatic mutations outside the SH2-domain of STAT3 in LGL leukemia

E Andersson et al. Leukemia. 2016 May.
No abstract available

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Conflict of interest statement

Disclosure of conflicts of interest

H.R. has received honoraria from Novartis. S.M. has received honoraria and research funding from Novartis, Pfizer and Bristol-Myers Squibb.

Figures

Figure 1
Figure 1. Sorting, vbeta and sequencing results from patient 1
A. The index patient is a 60-year-old male diagnosed with T-LGL leukemia in 2002 suffering from anemia, neutropenia, B-cell dyscrasia and hypergamma-globulinemia (Supplemental table S1). Mononuclear cells (MNCs) were separated from whole blood with Ficoll-Paque™ PLUS (GE Healthcare). The MNCs were then sorted by flow cytometry using markers for CD45+ (PerCP), CD3+ (FITC), CD4+ (APC) and CD8+ (PE) (Becton Dickinson). Based on flow cytometry the CD8+ tumor cells (90% of CD3+ population) and the CD4+ control cells (9% of the CD3+ population) were sorted. B. Peripheral blood from the patient was used to determine the TCR Vβ repertoire of human T lymphocytes with the IO Test® Beta Mark TCR Vβ Repertoire Kit (Beckman-Coulter Immunotech). Flow cytometry measurement of the TCR repertoire of patient 1 revealed a 95% Vb.17 clone in the CD8+ cells. C. Location of novel mutations in the coiled-coil alpha domain and DNA-binding domain in addition to the previously described SH2 domain mutations in STAT3. D. 3D visualization of the location of the variants F174S and H410R in STAT3 (Polyphen-2).
Figure 2
Figure 2. Functional characterization of the variants outside of the SH2-domain in STAT3
A. HEK cells harboring an sis-inducible (SIE) response element luciferase reporter were transfected with vector alone (empty), wild type STAT3 (wt), the three identified mutants (F174S, S381Y and H410R) or the most prevalently seen SH2-domain mutant (Y640F). The cells were either left unstimulated or treated for 3h with IL-6 to induce STAT3 phoshorylation and activation. Luciferase activity was determined with ONE-Glo™ Luciferase Assay System and results are reported as fold change (FC) compared to unstimulated wild type STAT3. Each condition was tested in triplicate and the statistical significance was calculated with Kruskal-Wallis and Dunn’s multiple comparison tests (*=p<0.05, ***=p<0.0001, error bars representing SD). B. To investigate the phosphorylation status of the variants, HEK-293 cells transfected with the above mentioned variants were analyzed by western blot with a phosphoSTAT3 specific antibody. Protein lysates of the different variants were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. STAT3 protein levels of the different variants were used to normalize for the transfection efficacy. β-actin was used as a loading control. C. Total RNA of the transfected HEK293 cells was extracted and converted to cDNA using random primers. Expression levels of six known STAT3 target genes (SOCS3, JAK2, MYC, JUNB, BCL3, CCL2) were measured and the results were normalized against four housekeeping genes (NONO, PGK-1, GAPDH, LDHA), which showed uniform expression across all samples. All reactions were run in triplicate wells and gene expression was quantified using the delta-delta Cq method.
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References

    1. Sokol L, Loughran TP., Jr Large granular lymphocyte leukemia. Current hematologic malignancy reports. 2007;2(4):278–82. - PubMed
    1. Koskela HL, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanmaki H, Andersson EI, et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. The New England journal of medicine. 2012;366(20):1905–13. - PMC - PubMed
    1. Fasan A, Kern W, Grossmann V, Haferlach C, Haferlach T, Schnittger S. STAT3 mutations are highly specific for large granular lymphocytic leukemia. Leukemia. 2013;27(7):1598–600. - PubMed
    1. Epling-Burnette PK, Liu JH, Catlett-Falcone R, Turkson J, Oshiro M, Kothapalli R, et al. Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. The Journal of clinical investigation. 2001;107(3):351–62. - PMC - PubMed
    1. Kucuk C, Jiang B, Hu X, Zhang W, Chan JK, Xiao W, et al. Activating mutations of STAT5B and STAT3 in lymphomas derived from gammadelta-T or NK cells. Nature communications. 2015;6:6025. - PMC - PubMed

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