Somatic STAT3 mutations in large granular lymphocytic leukemia

Abstract

Background: T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias.

Methods: We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes.

Results: Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations.

Conclusions: The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

Figure 1. Results of Flow Cytometry and Sequencing in the Index Patient

The index patient was a 70-year-old man with untreated large granular lymphocytic leukemia and grade 3 neutropenia. Panel A shows the results of flow cytometry. CD3 and CD16/56 staining of the lymphocyte population indicated that the majority of cells were CD3-positive and CD16/56-negative (left plot); 92% of CD3+ gated cells were CD4-negative and CD8-positive (middle plot). CD8+ T cells expressed T-cell receptor alpha and beta (right plot). APC denotes allophycocyanin, Cy7 cyanine 7, FITC fluorescein isothiocyanate, PE phycoerythrin, TCR ab T-cell receptor alpha and beta, and TCR gd gamma and delta. Panel B shows the results of Vβ and validation sequencing of the CD8 sorted cells. When the sample was obtained for exome-sequencing analysis, the patient had one large, predominantly T-cell clone: 94% of CD8+ cells consisted of a single Vβ16 clone (upper graph). A heterozygous STAT3 SH2 mutation T→A (protein D661V) was detected in CD8+ T cells (lower graph).

Figure 2. Locations of STAT3 Mutations and Crystal Structure of STAT3 Homodimer

All seven STAT3 mutations (D661V, D661Y, D661H, N647I, K658 N, Y640F, and Y657_K658insY) are found in the SH2 domain of the STAT3 protein and are only a couple of amino acids apart, as shown in a linear representation of the STAT3 primary sequence (Panel A). The structure of the STAT3 domain is represented according to the definition in the protein families database (Sanger Institute, http://pfam.sanger.ac.uk). A three-dimensional model of the STAT3 dimer (Panel B) shows that the mutated residues are located at the dimerization interface of the SH2 domain. Six STAT3 mutations are shown in one of the two subunits (magenta), as is the in-frame insertion mutation (yellow).

Figure 3. Immunohistochemical Staining of Bone Marrow–Biopsy Samples from a Healthy Control and from Three Patients with Large Granular Lymphocytic Leukemia

Panels A through D show paraffin sections from bone marrow–biopsy samples stained with monoclonal antibodies against phosphorylated STAT3 (pSTAT) (left) and CD57 (right). No staining is observed in the samples obtained from healthy controls (a representative example of which is shown in Panel A). The samples obtained from three patients who had large granular lymphocytic leukemia with STAT3 mutations (Panels B, C, and D) show infiltration by lymphocytes, which stain for pSTAT3 (in the nucleus) and for CD57 (in the cytoplasm). Panel E shows fractionated mononuclear cells from a healthy donor and from Patient 30 and Patient 1, who had Y640F and D661V STAT3 mutations, respectively. The mutant forms are hyperphos-phorylated and localized predominantly in the nucleus. Normalized aliquots of whole-cell (W), cytosolic (C), and nuclear (N) fractions were resolved on sodium dodecyl sulfate–polyacrylamide-gel electrophoresis and transferred to polyvinylidene fluoride membrane, and Western blot analysis was performed with the use of anti-pSTAT3, anti-STAT3, and anti-Erk1 antibodies. Erk1/2 denotes extracellular regulated kinases 1 and 2.

Figure 4. Quantification of STAT3-Responsive Genes in Patients with STAT3 Mutations, and Activation of STAT3-Dependent Transcription by Wild-Type and Mutant Protein Variants

Panel A shows the mean levels of RNA expression of STAT3-responsive genes from CD8 sorted cells obtained from 5 healthy controls, 8 patients with STAT3 mutations, and 10 patients without STAT3 mutations, with the use of the HumanHT-12 v4 Expression BeadChip Array (Illumina). I bars represent standard errors. One asterisk denotes P<0.05, two asterisks P<0.01, and three asterisks P<0.001. Panel B shows human embryonic kidney cells that were left untreated or treated with interleukin-6 to induce STAT3 phosphorylation and activation. These cells, which harbored a luciferase reporter gene under the control of a STAT3-responsive sis-inducible element (SIE), were transfected with vector alone, wild-type STAT3, or the two mutants most frequently identified (Y640F and D661V), and each condition was tested in triplicate.