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T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T-cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15, and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine-mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine-mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL, and other IL-2 or IL-15 driven hematopoietic malignancies.

Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T-cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15, and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine-mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine-mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL, and other IL-2 or IL-15 driven hematopoietic malignancies.

Failure to undergo activation-induced cell death due to global dysregulation of apoptosis is the pathogenic hallmark of large granular lymphocyte (LGL) leukemia. Consequently, immunosuppressive agents are rational choices for treatment. This first prospective trial in LGL leukemia was a multicenter, phase 2 clinical trial evaluating methotrexate at 10 mg/m2 orally weekly as initial therapy (Step 1). Patients failing methotrexate were eligible for treatment with cyclophosphamide at 100 mg orally daily (Step 2). The overall response in Step 1 was 38% with 95% confidence interval (CI): 26%, 53%. The overall response in Step 2 was 64% with 95% CI: 35%, 87%. The median overall survival for patients with anemia was 69 months with a 95% CI lower bound of 46 months and an upper bound not yet reached. The median overall survival for patients with neutropenia has not been reached 13 years from study activation. Serum biomarker studies confirmed the inflammatory milieu of LGL but were not a priori predictive of response. We identify a gene expression signature that correlates with response and may be STAT3 mutation driven. Immunosuppressive therapies have efficacy in LGL leukemia. Gene signature and mutational profiling may be an effective tool in determining whether methotrexate is appropriate therapy.

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P

Natural killer (NK) cell lymphomas/leukemias are rare neoplasms with an aggressive clinical behavior. The majority of the cases belong to extranodal NK/T-cell lymphoma, nasal type (ENKTL) in the current WHO classification scheme. Gene-expression profiling (GEP) of 21 ENKTL and NK-cell lymphoma/leukemia patients, 17 NK- and T-cell lines and 5 indolent NK-cell large-granular-lymphocytic proliferation was performed and compared with 125 peripheral T-cell lymphoma (PTCL) patients previously studied. The molecular classifier derived for ENKTL patients was comprised of 84 transcripts with the majority of them contributed by the neoplastic NK cells. The classifier also identified a set of gamma delta-PTCLs both in the ENKTL cases as well as in cases initially classified as PTCL-not otherwise specified. These gamma delta-PTCLs expressed transcripts associated with the T-cell receptor (TCR)/CD3 complex, suggesting T cell rather than NK-cell lineage. They were very similar to NK-cell tumors by GEP, but were distinct from cytotoxic (alpha beta)-PTCL and hepatosplenic T-cell lymphoma, indicating derivation from an ontogenically and functionally distinct subset of gamma delta T cells. They showed distinct expression of V gamma 9, V delta 2 transcripts and were positive for TCR gamma, but negative for TCR beta by immuno-histochemistry. Targeted inhibition of two oncogenic pathways (AURKA and NOTCH-1) by small-molecular inhibitors induced significant growth arrest in NK-cell lines, thus providing a rationale for clinical trials of these inhibitors in NK-cell malignancies. Leukemia (2011) 25, 348-358; doi:10.1038/leu.2010.255; published online 5 November 2010