Thomas P. Loughran

Somatic STAT3 mutations in large granular lymphocytic leukemia.

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.).

Leukemia of large granular lymphocytes: association with clonal chromosomal abnormalities and autoimmune neutropenia, thrombocytopenia, and hemolytic anemia

Three patients had leukocytosis of large granular lymphocytes and chronic neutropenia. Clonal chromosomal abnormalities (trisomy 8 and trisomy 14) and lymphocytic infiltration of splenic red pulp, hepatic sinusoids, and bone marrow indicated the neoplastic nature of the large granular lymphocytes. Demonstration of a T3+, T8+, HNK-1 + phenotype and low natural killer cell activity that was augmented by interferon treatment showed the leukemic cells to be immature natural killer cells. Multiple autoantibodies were present and included rheumatoid factor and antinuclear, antineutrophil, antiplatelet, and antierythrocyte antibodies, suggesting a defect of B-cell immunoregulation. In addition, in-vitro studies showed impaired suppression of immunoglobulin biosynthesis by abnormal cells from one patient. Antineutrophil antibodies and absence of direct cell-mediated inhibition of granulocyte-macrophage colony formation supported a humoral immune mechanism for the neutropenia. In these patients the syndrome of splenomegaly, multiple autoantibodies with neutropenia, and lymphocytosis of large granular lymphocytes is due to a neoplastic proliferation of immature natural killer cells.

Network model of survival signaling in large granular lymphocyte leukemia

T cell large granular lymphocyte (T-LGL) leukemia features a clonal expansion of antigen-primed, competent, cytotoxic T lymphocytes (CTL). To systematically understand signaling components that determine the survival of CTL in T-LGL leukemia, we constructed a T-LGL survival signaling network by integrating the signaling pathways involved in normal CTL activation and the known deregulations of survival signaling in leukemic T-LGL. This network was subsequently translated into a predictive, discrete, dynamic model. Our model suggests that the persistence of IL-15 and PDGF is sufficient to reproduce all known deregulations in leukemic T-LGL. This finding leads to the following predictions: (i) Inhibiting PDGF signaling induces apoptosis in leukemic T-LGL. (ii) Sphingosine kinase 1 and NFκB are essential for the long-term survival of CTL in T-LGL leukemia. (iii) NFκB functions downstream of PI3K and prevents apoptosis through maintaining the expression of myeloid cell leukemia sequence 1. (iv) T box expressed in T cells (T-bet) should be constitutively activated concurrently with NFκB activation to reproduce the leukemic T-LGL phenotype. We validated these predictions experimentally. Our study provides a model describing the signaling network involved in maintaining the long-term survival of competent CTL in humans. The model will be useful in identifying potential therapeutic targets for T-LGL leukemia and generating long-term competent CTL necessary for tumor and cancer vaccine development.

Large granular lymphocyte leukemia. Report of 38 cases and review of the literature.

LGL leukemia results from a chronic, clonal proliferation of LGL. Chronic neutropenia with recurrent bacterial infection and splenomegaly are common clinical manifestations. Rheumatoid arthritis coexists in some of these patients, who thus resemble patients with Felty syndrome. Other hematologic abnormalities that may occur include pure red-cell aplasia and adult-onset cyclic neutropenia. Lymphoid infiltration of bone marrow, splenic red pulp cords, and hepatic sinusoids is characteristic; lymph node and skin involvement are rare. Multiple serologic abnormalities are frequently present, including positive tests for rheumatoid factor and/or antinuclear antibody, polyclonal hypergammaglobulinemia, and circulating immune complexes. Antineutrophil and antiplatelet antibodies are often present. Leukemic LGL exhibit phenotypic heterogeneity; the most common phenotype in our patients is CD2+, CD3+, CD8+, HNK-1+, CD16-. Despite markedly increased numbers of LGL, functional activity of the cells is usually decreased. The mechanism of cytopenias is uncertain: in pure red-cell aplasia, it appears to be due to suppressive effect on erythropoiesis by abnormal LGL, but in patients with chronic neutropenia it may be antibody-mediated. Although most patients appear to have a relatively benign clinical course, mortality from infections and progressive lymphoproliferation is substantial. Optimal therapy remains undefined. Some preliminary evidence suggests that LGL leukemia may be associated with infection with a retrovirus similar to HTLV-I. Although relatively rare, LGL leukemia is of interest because a better understanding of this disease process may contribute to our knowledge of autoimmune diseases, the immunoregulatory functions of LGL, and the mechanisms controlling normal hematopoiesis.

STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Clinical features of large granular lymphocyte leukemia

The spectrum of large granular lymphocyte (LGL) proliferations consists of four distinct entities: reactive/transient LGL expansion, chronic LGL lymphocytosis, classical indolent LGL leukemia, and aggressive LGL leukemia. LGL leukemias are classified as lymphoid malignancies. They are divided into CD3(+)/T-cell LGL (85% of cases) and CD3(-)/natural killer (NK) cell LGL leukemia (15% of cases). Recent progress in the comprehension of the leukemogenesis has shown a dysregulation of survival signals in leukemic cells. Identification of LGL expansion has been improved using T-cell receptor (TCR)beta/gamma polymerase chain reaction (PCR) analysis and a combination of Vbeta and killer cell immunoglobulin-like receptor (KIR)-specific monoclonal antibodies. LGL leukemias are characterized by a clonal LGL infiltration of the bone marrow, spleen, and liver. Monoclonality is recognized by phenotypic, molecular, and karyotypic analysis. T-LGL leukemias affect the elderly and display a relatively indolent behavior. Approximately 60% to 70% of patients are symptomatic: recurrent infections secondary to chronic neutropenia, anemia, and autoimmune disease such as rheumatoid arthritis are the main clinical manifestations. Long-lasting remission can be obtained with low-dose methotrexate, cyclosporine A, or cyclophosphamide. Conversely, NK LGL leukemias behave aggressively, and most patients do not respond to chemotherapy.

Cooperative Regulation of Mcl-1 by Janus Kinase/STAT and Phosphatidylinositol 3-Kinase Contribute to Granulocyte-Macrophage Colony-Stimulating Factor-Delayed Apoptosis in Human Neutrophils

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

How I treat LGL leukemia

Large granular lymphocyte (LGL) leukemia is characterized by a clonal expansion of either CD3(+) cytotoxic T or CD3(-) NK cells. Prominent clinical features of T-LGL leukemia include neutropenia, anemia and rheumatoid arthritis (RA). The terminal effector memory phenotype (CD3(+)/CD45RA(+)/CD62L(-)CD57(+)) of T-LGL suggests a pivotal chronic antigen-driven immune response. LGL survival is then promoted by platelet-derived growth factor and interleukin-15, resulting in global dysregulation of apoptosis and resistance to normal pathways of activation-induced cell death. These pathogenic features explain why treatment of T-LGL leukemia is based on immunosuppressive therapy. The majority of these patients eventually need treatment because of severe or symptomatic neutropenia, anemia, or RA. No standard therapy has been established because of the absence of large prospective trials. The authors use low-dose methotrexate initially for T-LGL leukemia patients with neutropenia and/or RA. We recommend either methotrexate or oral cyclophosphamide as initial therapy for anemia. If treatment is not successful, patients are switched to either the other agent or cyclosporine. The majority of patients experience an indolent clinical course. Deaths infrequently occur because of infections related to severe neutropenia. As there are no curative therapeutic modalities for T-LGL leukemia, new treatment options are needed.

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR–ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr–Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr–Abl kinase. In this study, we show that blocking Bcr–Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr–Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr–Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr–Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr–Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.