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Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher’s exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch’s t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Large granular lymphocyte (LGL) leukemia is a rare hematological disorder with expansion of the T‐cell or natural killer (NK) cell lineage. Signal transducer and activator of transcription 3 (STAT3) exhibits somatic activating mutations in 30%‐40% of LGL leukemia cases. Transcriptional targets of STAT3 include inflammatory cytokines, thus previous studies have measured cytokine levels of LGL leukemia patients compared to normal donors. Sphingolipid metabolism is a growing area of cancer research, with efforts focused on drug discovery. To date, no studies have examined serum sphingolipids in LGL leukemia patients, and only one study compared a subset of cytokines between the T‐LGL and NK‐LGL subtypes. Therefore, here, we included both LGL leukemia subtypes with the goals of (a) measuring serum sphingolipids for the first time, (b) measuring cytokines to find distinctions between the subtypes, and (c) establishing relationships with STAT3 mutations and clinical data. The serum analyses identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) significantly different in the LGL leukemia group compared to normal donors. In a mixed STAT3 mutation group, D661Y samples exhibited the highest mean corpuscular volume (MCV) values. We explored this further by expanding the cohort to include larger groups of single STAT3 mutations. Male D661Y STAT3 samples had lower Hgb and higher MCV compared to wild type (WT) or Y640F counterparts. This is the first report examining large groups of individual STAT3 mutations. Overall, our results revealed novel serum biomarkers and evidence that D661Y mutation may show different clinical manifestation compared to WT or Y640F STAT3., Analysis of LGL leukemia serum identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) that were significantly different compared to normal donors. Comparing three groups of STAT3 mutation status (WT, Y640F, D661Y) in LGL leukemia patients showed the D661Y mutation group had higher mean corpuscular volume and lower hemoglobin compared to the other groups.

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16

Context: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a subset of large granular lymphocyte (LGL) leukemia included as a provisional entity in the 2016 WHO classification of lymphoid neoplasms, which is characterized by clonal expansion of NK cells with no standardized therapy. STAT3 and TET2 mutations were previously each reported in 15%–30% of cases; however, the genomic basis of the remaining cases is still unknown. Objective: We aimed to perform a comprehensive genomic and transcriptomic analysis of CLPD-NK to elucidate the genomic landscape, investigate mechanisms of pathogenesis, and improve diagnostic classification and treatment strategies. Methods: CLPD-NK samples (n=121) were examined by whole-genome, targeted, and/or whole-transcriptome sequencing. Structural modeling, G-protein signaling, β-arrestin signaling, G-protein-coupled receptor (GPCR) for CCL22, CCR4 internalization, and chemotaxis were examined. Cell line-derived xenografts were used to examine cell proliferation and phenotype. Results: Somatic mutations in the chemokine CCL22 were found in 21.5% of CLPD-NK patients but not in 2837 cases of other hematological malignancies, including T-LGL and other NK leukemias. Clinically, there was a trend to an association with cutaneous complications and/or neurological symptoms. CCL22 mutations were enriched at the highly conserved residues Leu45, Pro46, and Pro79 and were mutually exclusive of STAT3 mutations. CCL22 mutations were associated with dysregulated gene expression, similar to normal CD56bright NK cells, with enrichment for activation of cytokine signaling pathways, representing a distinct subgroup. Most CCL22 mutations were substitutions to Arg, a basic residue, and Leu45Arg was predicted to promote electrostatic interactions with CCR4-Tyr22/Glu23 acidic residues. The mutations resulted in ligand-biased CCR4 signaling, in which activation of G-protein signaling was unchanged, but recruitment of β-arrestin, which mediates receptor internalization, was impaired, which, in turn, resulted in enhanced cell chemotaxis. CCL22 mutations drove enhanced cell proliferation in vivo in hIL-15-transgenic mice and induced an expression profile resembling that of primary samples, indicating the importance of microenvironmental crosstalk in pathogenesis. Conclusions: Somatic CCL22 mutations defined a new subgroup of CLPD-NK similar to CD56bright normal NK cells and represented a novel mechanism of leukemogenesis in which gain-of-function chemokine mutations drive tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

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.

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by clonal expansion of either CD3+ cytotoxic T cells expressing T cell receptor (TCR) alpha beta or CD3- natural killer (NK) cells. A less frequent CD3+ T cell subtype expresses TCR gamma delta (GD). Here, we present the molecular landscape of known LGL types (T, NK, GD) from analysis of the largest patient cohort assembled to date. An integrative analysis of genomic datasets from all LGL subtypes is necessary to more precisely define the shared and unique etiology of this rare disorder. Methods: We collected paired saliva and PBMC samples and related clinical information from 116 LGL leukemia patients after informed consent. The assembled cohort consisted of 93 T-LGL, 11 NK-LGL, and 12 GD T-LGL patients. Genomic analyses were performed on the leukemic (PBMC) and germline (saliva) samples after whole exome sequencing (WES) and transcriptome sequencing (RNAseq, PBMC only). Results: Somatic mutations were detected in the previously described potential drivers STAT3 (n=56), TNFAIP3 (n=9), and PIK3R1 (n=4). We also identified somatic mutations in CDH8 (n=3) and CCL22 (n=4), which we postulate as putative drivers based on mutational clustering. CDH8 was mutated in all three LGL subtypes, but CCL22 somatic mutations were only observed in NK-LGL patients. We observed that STAT3 and CCL22 together account for 64% (7/11) of NK-LGL cases. STAT3 is the most recurrently mutated gene in LGL leukemia, yet concurrent molecular and clinical features are incompletely defined. Interestingly, patients with STAT3 mutations have a higher mutational burden (P=0.0006) compared to those with wild-type (WT) STAT3. This effect is independent of the age of the patient, which correlates with the mutational burden (R=0.26, P=0.0039) and agrees with the finding that the dominant mutational signatures in this cohort exhibit clock-like properties. We also observed that patients with STAT3 mutations are enriched (P=0.0273) for additional mutations in chromatin modifier enzymes such as KMT2D, TET2, DNMT3A, and SETD1B (Figure 1). We found that ~10% of the samples exhibit broad somatic copy-number aberrations, and a patient with somatic mutations in STAT3 and KMT2D displayed high-level microsatellite instability. STAT3 mutations were also significantly associated with increased expression of genes involved in apoptosis, complement activation, and interferon cytokine signaling compared to STAT3 WT (FDR < 0.05). Early-onset LGL patients with age 51 years or less (n=28), as defined by the bottom quartile of the cohort, displayed no differential enrichment of the somatic driver genes. Interestingly, the age of the patient was significantly associated with absolute neutrophil counts (ANC) (P = 0.0068), with younger patients exhibiting lower neutrophil counts, even after adjusting for the presence of STAT3 mutation, as it is associated with lower ANC. As neutropenia is a hallmark feature of LGL leukemia and often a trigger for initiating therapy, the association of young age with lower neutrophil counts and lower somatic mutational burden suggests other mechanisms may be involved. Focusing on the germline variants, we found that 17 of the patients (14.6%) had at least one pathogenic or likely pathogenic germline variant with known oncogenic association as annotated using CharGer. 5 patients had pathogenic mutations in known tumor suppressors including FANCC (n=1), BRCA1 (n=1), PALB2 (n=1), MUTYH (n=1), and SDHA (n=1), while 1 patient had pathogenic mutations in ALK, a known oncogene. Conclusions: We report on the genomic analyses done on whole exome and RNA-seq data from the largest cohort assembled for LGL leukemia to date. We show that the presence of STAT3 mutation is significantly associated with an increase in mutation burden and additional somatic mutations in chromatin modifiers, hinting at potential pathogenic mechanisms within STAT3 mutated patients. By combining LGL subtypes in our analysis, we were able to identify CDH8 as a putative driver that is present in T, NK, and GD subtypes. Additionally, we report CCL22 mutations specific to NK-LGL leukemia, however did not detect any subtype specific mutations in GD T-LGL. We found that about 15% of the patients carry at least one pathogenic germline variant with known oncogenic associations. These findings highlight emerging etiologic insights into this rare disorder. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees.

Genomic analysis of cancer offers the hope of identifying new treatments or aiding in the selection of existing treatments. Rare leukemias pose additional challenges in this regard as samples may be hard to acquire and when found the underlying pathway may not be attractive to drug development since so few individuals are affected. In this case, it can be useful to identify common mutational overlap among subsets of rare leukemias to increase the number of individuals that may benefit from a targeted therapy. This chapter examines the current mutational landscape of large granular lymphocyte (LGL) leukemia with a focus on STAT3 mutations, the most common mutation in LGL leukemia to date. We examined the linkage between these mutations and autoimmune symptoms and disorders, in cases of obvious and suspected LGL leukemia. We then summarized and compared mutations in a set of other rare leukemias that also have JAK/STAT signaling pathway activation brought about by genomic changes. These include T-cell acute lymphoblastic leukemia (T-ALL), T-cell prolymphocytic leukemia (T-PLL), cutaneous T-cell lymphoma (CTCL), select peripheral T-cell lymphoma (PTCL), and adult T-cell leukemia/lymphoma (ATLL). Though STAT3 activation is common in these leukemias, the way in which it is achieved, such as the activating cytokine pathway and/or the co-mutational background, is quite diverse.

BackgroundLarge granular lymphocyte (LGL) leukemia is an uncommon cancer characterized by a sustained clonal proliferation of LGL cells. Antibodies reactive to retroviruses have been documented in the serum of patients with LGL leukemia. Culture or molecular approaches have to date not been successful in identifying a retrovirus.MethodsBecause a retrovirus must integrate into the genome of an infected cell, we focused our efforts on detecting a novel retrovirus integration site in the clonally expanded LGL cells. We present a new computational tool that uses long-insert mate pair sequence data to search the genome of LGL leukemia cells for retrovirus integration sites. We also utilize recently published methods to interrogate the status of polymorphic human endogenous retrovirus type K (HERV-K) provirus in patient genomes.ResultsWhile our analysis did not reveal any new retrovirus insertions in LGL genomes from LGL leukemia patients, we did identify four HERV-K provirus integration sites that are polymorphic in the human population and absent from the human reference genome, hg19. To determine if the prevalence of these or other polymorphic proviral HERV-Ks differed between LGL leukemia patients and the general population, we applied a recently developed approach that reports all sites in the human genome occupied by a proviral HERV-K. Using the 1000 genomes project (KGP) data as a reference database for HERV-K proviral prevalence at each polymorphic site, we show that there are significant differences in the number of polymorphic HERV-Ks in the genomes of LGL leukemia patients of European origin compared to individuals with European ancestry in the KGP data.ConclusionsOur study confirms that the integration of a new infectious or endogenous retrovirus does not cause the clonal expansion of LGL cells in LGL leukemia, although we do not rule out that these cells could be responding to retroviral antigens produced in other cell types. However, it is of interest that the burden of polymorphic proviral HERV-K is elevated in LGL leukemia patient genomes. Our research emphasizes the merits of comprehensive genomic assessment of HERV-K in cancer samples and suggests that further analyses to determine contributions of HERV-K to LGL leukemia are warranted.

Commonly known for its critical role in calcium homeostasis and bone mineralization, more recently vitamin D has been implicated in haematological cancer pathogenesis and shows promise as an anti-cancer therapy. Serum levels of 25(OH)D3, the precursor to the active form of vitamin D, calcitriol, are typically lower in patients with haematological disease compared to healthy individuals. This often correlates with worse disease outcome. Furthermore, diseased cells typically highly express the vitamin D receptor (VDR), which is required for many of the anti-cancer effects observed in multiple in vivo and in vitro cancer models. In abnormal haematological cells, vitamin D supplementation promotes apoptosis, induces differentiation, inhibits proliferation, sensitizes tumor cells to other anti-cancer therapies, and reduces the production of pro-inflammatory cytokines. Although the dosage of vitamin D required to achieve these effects may induce hypercalcemia in humans, analogs and combinatorial treatments have been developed to circumvent this side effect. Vitamin D and its analogs are well tolerated in clinical trials and thus further investigation into the use of these agents in the clinic is warranted. Here we review the current literature in this field.

Large granular lymphocyte leukemia (LGLL) is a rare incurable chronic disease typically characterized by clonal expansion of CD3+ cytotoxic T-cells. Two signal transducer and activator of transcription factors, STAT1 and STAT3, are constitutively active in T-LGLL. Disruption of this activation induces apoptosis in T-LGLL cells. Therefore, considerable efforts are focused on developing treatments that inhibit STAT activation. Calcitriol, the active form of vitamin D, has been shown to decrease STAT1 and STAT3 phosphorylation in cancer cell lines and autoimmune disease mouse models. Thus, we investigated whether calcitriol could be a valid therapeutic for T-LGLL. Calcitriol treatment of the TL-1 cell line (model of T-LGLL) led to decreased phospho-Y701 STAT1 and phospho-Y705 STAT3 and increased vitamin D receptor (VDR) levels. Doses of 10 and 100 nM calcitriol also significantly decreased the inflammatory cytokine IFN-γ in the TL-1 cell line. The overall cell viability did not change when the TL-1 cell line was treated with 0.1 to 1000 nM calcitriol. Studies with primary T-LGLL patient peripheral blood mononuclear cells showed that the majority of T-LGLL patients have detectable VDR and activated STATs in contrast to normal donor controls. Treatment of primary T-LGLL patient cells with calcitriol recapitulated findings from the TL-1 cell line. Overall, our results suggest that calcitriol may reprogram T-cells to decrease essential STAT activation and pro-inflammatory cytokine output. These data support further investigation into calcitriol as an experimental therapeutic for T-LGLL.

Background: The pathogenesis of chronic lymphoproliferative disorder of NK-cells (CLPD-NK) is poorly understood. Mutations in the JAK/STAT pathway (especially STAT3) are found in 30% of patients, but the genetic or exogenous drivers responsible for other cases are unknown. Here we comprehensively define the genetic drivers of CLPD-NK by integrated genome and transcriptome sequencing (WGS/WTS) of a large cohort of cases and complementary functional analyses. Methods: We studied 63 CLPD-NK patients (M/F: 42/21, median age: 71 years [35-89 y]) by WGS, WTS and flow cytometry. A validation cohort of 67 patients (M/F: 43/24, median age: 64 years [7-91]) was analyzed by targeted sequencing. To study the role of CCL22 in CLPD-NK pathogenesis, we examined internalization of the CCL22 receptor, CCR4, and cell chemotaxis in response to exogenous wild type (wtCCL22) or mutant CCL22 (mtCCL22: L45R, P79R, IL87_88NF) in CCR4-expressing Ba/F3 cells (Ba/F3-CCR4). To examine potential autocrine/paracrine activity, we exchanged supernatants of Ba/F3-CCR4-wtCCL22 and -mtCCL22 cells and examined CCR4 expression. To examine the in vivo effects of the mutations on proliferation and phenotype, GFP-tagged empty vector, wtCCL22, or mtCCL22-transduced NK-92 cells were engrafted into IL15-transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG) mice. Human NK-92 cells isolated from spleens of moribund mice were analyzed by WTS and immunophenotyping. Results: WGS of 63 CLPD-NK identified STAT3 mutations in 18 (29%) cases, with mutually exclusive CCL22 mutations (mtCCL22) in 14 (22%) patients. WGS of 4500 hematological malignancies showed that mtCCL22 were only found in CLPD-NK. Recurrent co-mutations in both groups were found in ATM (n=3), FAS (n=2) and TET2 (n=5). Of the remaining patients, 23/31 had one or more mutation including epigenetic regulators (n=12), signaling components (n=7) or TP53 (n=4). Our findings of CCL22 mutations were confirmed in an independent validation cohort with STAT3 mutations in 19/67 (28%) and mtCCL22 in 13/67 (19%). CCL22 mutations were clustered at the conserved leucine 45 and proline 79 residues (Fig. 1A). Sequencing of purified CD3+ T and CD56+ NK cells showed that the mtCCL22 were somatic mutations acquired by the CD56+ NK population. mtCCL22 defined a subgroup of CLPD-NK, with high NCAM1 (CD56) positivity by flow cytometry (p 1.3, adj P CCL22 induces chemotaxis of CCR4-expressing Th2 T cells, and also drives CCR4 internalization mediated by G-protein coupled receptor signaling via β-arrestin as a negative feedback mechanism. All three tested mtCCL22 resulted in significant and prolonged attenuation of CCR4 internalization compared to wtCCL22 (Fig 1C). Exchanging the supernatant of mtCCL22-treated Ba/F3-CCR4 cells for that from wtCCL22-treated cells decreased CCR4 expression, and vice versa, suggesting that mtCCL22 regulates CCR4 surface expression and signaling through an autocrine or paracrine mechanism (Fig 1D). Furthermore, mtCCL22 increased chemotaxis of CCR4-Ba/F3 cells in a transwell cell migration assay compared to wtCCL22 (Fig 1E). Expression of mtCCL22 enhanced engraftment of NK-92 cells which exhibited higher XCR1,GZMK and surface CD56 expression compared to controls, as well as deregulation of type I interferon response pathways indicating reactive cytokine production similar to that observed in CLPD-NK patients. Conclusions: mtCCL22 were detected in 19-22% of CLPD-NK, were mutually exclusive with STAT3 mutations, and define a distinct subgroup of CLPD-NK with genetic features that recapitulate CD56bright cytokine-producing NK cells. We show that mtCCL22 enhanced cellular chemotaxis by preventing CCR4 internalization and promoted expansion of CD56+ NK cells in vivo. We recommend to study mtCCL22 in cases suspected of CLPD-NK, in order to distinguish clonal NK neoplasms from polyclonal reactive expansion. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Mullighan:Pfizer: Honoraria, Research Funding, Speakers Bureau; Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding.

Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100 nM EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (n = 8) vs. age- and sex-matched normal healthy donors (n = 8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NK-LGLL patients.

Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3(+) cytotoxic T lymphocytes or CD3(−) natural killer (NK) cells. Chronic antigen stimulation is postulated to promote long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, resulting in global dysregulation of apoptosis and resistance to activation-induced cell death. We reported previously that nuclear factor κB (NF-κB) is a central regulator of the survival network for leukemic LGL. However, the mechanisms that trigger constitutive activation of NF-κB in LGL leukemia remain undefined. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in tumor cells but can also activate NF-κB through interaction with TRAIL receptors 1, 2, and 4 (also known as DR4, DR5, and DcR2, respectively). The role of TRAIL has not been studied in LGL leukemia. In this study, we hypothesized that TRAIL interaction with DcR2 contributes to NF-κB activation in LGL leukemia. We observed upregulated TRAIL messenger RNA and protein expression in LGL leukemia cells with elevated levels of soluble TRAIL protein in LGL leukemia patient sera. We also found that DcR2 is the predominant TRAIL receptor in LGL leukemia cells. We demonstrated that TRAIL-induced activation of DcR2 led to increased NF-κB activation in leukemic LGL. Conversely, interruption of TRAIL-DcR2 signaling led to decreased NF-κB activation. Finally, a potential therapeutic application of proteasome inhibitors (bortezomib and ixazomib), which are known to inhibit NF-κB, was identified through their ability to decrease proliferation and increase apoptosis in LGL leukemia cell lines and primary patient cells.

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.

Large granular lymphocyte (LGL) leukemia is a chronic clonal lymphoproliferative disorder. Here, a T-LGL leukemia patient developed NK-LGL leukemia with residual leukemic T-LGL. TCRVβ usage and CDR3 sequence drifts were observed with disease progression. A STAT3 S614R mutation was identified in NK but not T-cells in the mixed leukemic stage. Multiple, non-dominant T-cell clones with distinct STAT3 mutations were present throughout. Our results suggest that T and NK-LGL leukemia may share common pathogenesis mechanisms and that STAT3 mutation alone is insufficient to bring about clonal expansion. Mutational and immunological monitoring may provide diagnostic and therapeutic significance in LGL leukemia., Highlights • Coexistence of NK and T cell clones in LGL leukemia. • Demonstration for the first time of a shift from T-LGL to NK type of LGL leukemia. • Emergence of a dominant STAT3-mutated clone in NK cells during disease progression. • Presence of additional STAT3-mutated clones that fail to become dominant over time.

Constitutively activated STAT1 and elevated IFN-γ are both characteristic of T cell large granular lymphocytic leukemia (T-LGLL), a rare incurable leukemia with clonal expansion of cytotoxic T cells due to defective apoptosis. Interferon gamma (IFN-γ) is an inflammatory cytokine that correlates with worse progression and symptomology in multiple autoimmune diseases and cancers. In canonical IFN-γ-STAT1 signaling, IFN-γ activates STAT1, a transcription factor, via phosphorylation of tyrosine residue 701 (p-STAT1). p-STAT1 then promotes transcription of IFN-γ, creating a positive feedback loop. We previously found that calcitriol treatment of the TL-1 cell line, a model of T-LGLL, significantly decreased IFN-γ secretion and p-STAT1 while increasing the vitamin D receptor (VDR) protein. Here we further explore these observations. Using TL-1 cells, IFN-γ decreased starting at 4 h following calcitriol treatment, with a reduction in the intracellular and secreted protein levels as well as the mRNA content. A similar reduction in IFN-γ transcript levels was observed in primary T-LGLL patient peripheral blood mononuclear cells (PBMCs). p-STAT1 inhibition followed a similar temporal pattern and VDR upregulation inversely correlated with IFN-γ levels. Using EB1089 and 25(OH)D(3), which have high or low affinity for VDR, respectively, we found that the decrease in IFN-γ correlated with the ability of EB1089, but not 25(OH)D(3), to upregulate VDR. However, both compounds inhibited p-STAT1; thus the reduction of p-STAT1 is not solely responsible for IFN-γ inhibition. Conversely, cells treated with VDR siRNA exhibited decreased basal IFN-γ production upon VDR knockdown in a dose-dependent manner. Calcitriol treatment upregulated VDR and decreased IFN-γ regardless of initial VDR knockdown efficiency, strengthening the connection between VDR upregulation and IFN-γ reduction. Our findings suggest multiple opportunities to further explore the clinical relevance of the vitamin D pathway and the potential role for vitamin D supplementation in T-LGLL.

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.

Y2Si2O7:Nd3+ nanofibers were synthesized for the first time through a combination of sol–gel and electrospinning methods. Annealing effects on the Y2Si2O7 polymorph transformation and fiber morphology were examined under a scanning electron microscope. About 901, 1061, and 1337 nm emission bands were observed in the ultrafine nanofibers, while the rough fibers with pinholes and a larger grain size exhibited only slight photoluminescence. The one-dimensional surface stress and low-temperature crystalline distortion shifted the emission bands of the Nd3+- doped Y2Si2O7 fibers.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

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.

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.

Src kinase has been linked to increased motility in the progression and metastasis of human colon cancer, although the mechanisms are not fully understood. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. This study examines the link between Src and integrin activity in the metastatic process in colon cancer cells. To determine Src involvement in integrin expression, the human colon cancer cell line, HCT116, was transfected with an activated Src construct and assayed for its ability to attach to and migrate across collagen and laminin. These cells attached more readily and migrated less rapidly on the extracellular matrix (ECM) than did cells transfected with empty vector. Examination of integrin levels showed a decrease in the alpha3 subunit in Src transfected cells as well as decreased cell surface localization of alpha3 integrin. The downregulation of alpha3 integrin was reversed by inhibition of Src and by inhibition of MAP kinase. Inhibition of alpha3 integrin using shRNA resulted in decreased MMP7 secretion, a possible cause of decreased invasion with low alpha3 integrin expression. This study shows that Src overexpression downregulates alpha3 integrin total protein expression and localization to the cell surface of HCT116 colon cancer cells. This indicates that Src activity may enhance metastasis by altering alpha3 integrin expression.

Background The discovery and mapping of genomic variants is an essential step in most analysis done using sequencing reads. There are a number of mature software packages and associated pipelines that can identify single nucleotide polymorphisms (SNPs) with a high degree of concordance. However, the same cannot be said for tools that are used to identify the other types of variants. Indels represent the second most frequent class of variants in the human genome, after single nucleotide polymorphisms. The reliable detection of indels is still a challenging problem, especially for variants that are longer than a few bases. Results We have developed a set of algorithms and heuristics collectively called indelMINER to identify indels from whole genome resequencing datasets using paired-end reads. indelMINER uses a split-read approach to identify the precise breakpoints for indels of size less than a user specified threshold, and supplements that with a paired-end approach to identify larger variants that are frequently missed with the split-read approach. We use simulated and real datasets to show that an implementation of the algorithm performs favorably when compared to several existing tools. Conclusions indelMINER can be used effectively to identify indels in whole-genome resequencing projects. The output is provided in the VCF format along with additional information about the variant, including information about its presence or absence in another sample. The source code and documentation for indelMINER can be freely downloaded from www.bx.psu.edu/miller_lab/indelMINER.tar.gz. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0483-6) contains supplementary material, which is available to authorized users.

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.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.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.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.).

BackgroundDiminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted.ResultsColon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NFκB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified ~700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to containcis-binding sequences for NFκB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NFκB occupancy at the promoter of one particular network geneDROSHA, encoding a microRNA processing enzyme. The resulting down-regulation ofDROSHAwas associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown ofDROSHAexpression in native HT29 cells increased miR-34a expression and 5-FU sensitivity.ConclusionOur findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

Cultured cell lines have played an integral role in the study of tumor biology since the early 1900's. The purpose of this study is to evaluate colorectal cancer (CRC) cell lines with respect to progenitor tumors and assess whether these cells accurately and reliably represent the cancers from which they are derived. Primary cancer cell lines were derived from fresh CRC tissue. Tumorigenicity of cell lines was assessed by subcutaneous injection of cells into athymic mice and calculation of tumor volume after 3 weeks. DNA ploidy was evaluated by flow cytometry. Invasive ability of the lines was tested with the MATRIGEL™ invasion assay at 24 or 48 hours. Cells were assessed for the presence of Kirsten-Ras (K-Ras), p-53, deleted in colon cancer (DCC), and Src. Protein profiling of cells and tissue was performed by surface enhanced laser desorption/ionization-time of flight/mass spectroscopy. microRNA expression in cell and tumor tissue samples was evaluated by FlexmiR™ MicroRNA Assays. Four cell lines were generated from tumor tissue from patients with CRC and confirmed to be tumorigenic (mean tumor volume 158.46 mm3). Two cell lines were noted to be diploid; two were aneuploid. All cell lines invaded the MATRIGEL™ starting as early as 24 hours. K-Ras, p53, DCC, and Src expression were markedly different between cell lines and corresponding tissue. Protein profiling yielded weak-to-moderate correlations between cell samples and respective tissues of origin. Weak-to-moderate tau correlations for levels of expression of human microRNAs were found between cells and respective tissue samples for each of the four pairings. Although our primary CRC cell lines vary in their expression of several tumor markers and known microRNAs from their respective progenitor tumor tissue, they do not statistically differ in overall profiles. Characteristics are retained that deem these cell lines appropriate models of disease; however, data acquired through the use of cell culture may not always be a completely reliable representation of tumor activity in vivo.

T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene.

Background Intra-species genetic variation can be used to investigate population structure, selection, and gene flow in non-model vertebrates; and due to the plummeting costs for genome sequencing, it is now possible for small labs to obtain full-genome variation data from their species of interest. However, those labs may not have easy access to, and familiarity with, computational tools to analyze those data. Results We have created a suite of tools for the Galaxy web server aimed at handling nucleotide and amino-acid polymorphisms discovered by full-genome sequencing of several individuals of the same species, or using a SNP genotyping microarray. In addition to providing user-friendly tools, a main goal is to make published analyses reproducible. While most of the examples discussed in this paper deal with nuclear-genome diversity in non-human vertebrates, we also illustrate the application of the tools to fungal genomes, human biomedical data, and mitochondrial sequences. Conclusions This project illustrates that a small group can design, implement, test, document, and distribute a Galaxy tool collection to meet the needs of a particular community of biologists.

Abstract 1302 Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T-cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene (Koskela et al, NEJM, 2012). In the remaining T-LGL patients, the pathogenetic mutations are not known. Methods: To identify additional somatic mutations, we chose two STAT3 mutation negative T-LGL leukemia patients for exome sequencing. CD8+ T-cells were used as test cells and matched CD4+ T-cells as control. The exome was captured with the Nimblegen SeqCap EZ Exome Library v2.0 and the sequencing was performed with the Illumina HiSeq2000 sequencing platform. Candidate somatic mutations were identified with a bioinformatics pipeline consisting of BWA for sequence alignment, Samtools for alignment filtering and Varscan for somatic mutation calling. Results: Index patient 1 was diagnosed with T-LGL leukemia at the age of 70 and a TCR repertoire assay revealed one minor T- cell clone in the leukemic sample (Vβ7.1: 28 %). Exome sequencing revealed 10 nonsynonymous nucleotide variants with p-values lower than 0.01, of which the tumor suppressor gene Protein tyrosine phosphatase (PTP) receptor T (PTPRT) had a variant frequency of 14%. PTPRT has previously been found to reverse Tyr705 phosphorylation on STAT3, a modification associated with STAT3 deactivation. In this novel mutation, a highly conserved hydrophobic valine residue is converted into methionine (V995M). The mutation occurs in the cytoplasmic part of the protein, within the tyrosine-protein phosphatase 1 domain. The PTPRT V995M mutation may thereby affect STAT3 activity by reducing dephosphorylation of Tyr705, thus increasing the expression of STAT3 target genes. Index patient 2 was a 40 year-old male with untreated T-LGL leukemia. A TCR repertoire assay showed one predominant T-cell clone in the leukemic T-cells (Vβ13.2: 70%). Exome sequencing revealed 8 nonsynonymous nucleotide variants with p-values lower than 0.01. The missense mutation K436E in Angiopoietin-2 (ANGPT2), presenting with the lowest somatic p-value (1,06−09) and highest variant frequency (34%), was the most relevant candidate involved in the pathogenesis of leukemia. The mutation occurs on the surface of ANGPT2 within the well-conserved fibrinogen C-terminal domain. This domain binds the receptor TIE2 and the change in the polarity induced by K436E mutation is likely to affect the binding of TIE2 by ANGPT2. Overexpression of ANGPT2 has previously been shown to confer an adverse prognostic factor in other forms of leukemia. While these mutations appear biologically relevant and exciting, we have not yet seen them in other LGL patients screened so far (n=80). Conclusions: Somatic mutations in the PTPRT and ANGPT2 genes may represent rare genetic causes for T-LGL leukemia. Screening for these mutations in a larger cohort of patients is warranted. The mutation in the PTPRT gene is particularly exciting as it may directly impact the STAT3 pathway, which is a common pathogenetic event in T-LGL leukemia. Inactivating mutations of the PTPRT gene may have the same functional consequence as activating mutations of STAT3 in LGL patients. Disclosures: Koskela: Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Kallioniemi:TEKES-FiDiPro: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

2-({4-[4-(acridin-9-ylamino)phenylthio]phenyl}(2-hydroxyethyl)amino)ethan-1-ol (CK0402) was selected as potential anticancer agent among the series of sulfur-containing 9-aminoacridine analogs previously synthesized by our group. CK0402 is a topoisomerase II inhibitor and has been shown to exert anti-cancer activities in both in vitro and in vivo assays. Our previous studies demonstrated that CK0402 exhibited highest activity in SKBR-3 cells among a panel of established human breast cancer cell lines; moreover, CK0402-induced responses of G2/M arrest, apoptosis and autophagy were stronger in ER-(−)/HER2-(+) SKBR-3 cells than in ER-(+)/HER2-(−) MCF-7 cells. These findings suggest that CK0402 may be effective against HER-2 (+) breast cancer. The objective of this study is to identify molecular makers in response to CK0402 treatment in HER-2 (+) cells. These treatment markers may be important for clinical application since they can be used to avoid lethal toxic side effects or unnecessary treatment to insensitive patients. To this end, we analyzed the global protein expression modulated by CK0402 treatment in SKBR-3 cells using LC-MS/MS with a quantitative MS-based iTRAQ labeling method. SKBR-3 cells were exposed to various concentrations of CK0402 for 24 and 72 hrs; in which, 2 plates of cells received control vehicle, 3 plates of cells received 1 μmol/L, and another 3 plates of cells received 5 μmol/L of CK0402. The cell lysates were denatured, reduced, alkylated, and then digested with trypsin. The resulting peptides were labeled with 8-plex iTRAQ™ reagents, fractionated by cation exchange, and subjected to a LC-MS/MS analysis. The levels of differentially expressed proteins between control and treated sample were compared using student's t-test. Selected proteins were then validated by Western blot analysis. We found that that a total of 219 proteins were differentially expressed in SKBR-3 cells (p < 0.05) after treated with 5 μmol/L of CK0402 for 72 hours. Among those, the levels of S100 proteins such as A7 and A8/A9 were increased 4.7- and 3.2-fold, respectively; in addition, the increases of these proteins were in a dose- and time- dependent manner, which is consistent with the growth inhibition effect of CK0402 in SKBR-3 cells. These results suggest that the S100 A7 and A8/A9 proteins are responders of CK0402 treatment in SKBR-3 cells. Current mechanistic studies are focusing on determining the roles of these identified proteins in the anticancer activities or toxic side effects of CK0402. The S100 proteins together with other proteins identified in this study could be used as effective indicators of the clinical response of patients to CK0402 treatment in future clinical studies. : Support: Susan G. Koman Breast Cancer Foundation Grant BCTR0707876 and Penn State HMC Dean's Feasibility Grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4576.

Organic carbon eroded and oxidized from marine sediments on the passive and active neo-Tethyan margins during the early Indian-Asian collision may have been sufficient to shift the carbon isotopic ratios of late Paleocene–early Eocene marine carbonates toward lighter values, and may have contributed to coeval global warming via the greenhouse effect. New limits on the timing of collision and the organic carbon content of Himalayan sedimentary sources and sinks allow us to estimate the net Paleogene flux. Our calculations suggest that continental collisions play a fundamental role in the global carbon cycle and climate through the exhumation as well as burial of organic carbon.

Abstract 871 Background: Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative disease, characterized by the clonal expansion of cytotoxic CD3+CD8+ T-cells or CD3-CD16/56+ natural killer (NK)-cells. It is often associated with autoimmune phenomena (e.g. cytopenias, rheumatoid arthritis). We recently identified somatic mutations in the STAT3 gene in 40% of monoclonal T-LGL cases (Koskela et al, NEJM, 2012). Here, we report the discovery and functional analysis of novel STAT5b mutations as well as small subclones of STAT3 mutations in other LGL patients, expanding the evidence implicating STAT activation in LGL. METHODS: In order to find novel LGL-leukemia associated mutations, exome sequencing was done from untreated STAT3 mutation negative T-LGL leukemia patients using CD8+ LGL-leukemic cells and matched CD4+ control cells. Samples from 158 T-LGL and 40 NK-LGL leukemia patients were further analyzed using both targeted Sanger sequencing and ultra-deep targeted next-gen amplicon sequencing with up to 10,000x coverage (MiSeq, Illumina). Functional analysis of mutated proteins was carried out in Hela cells by Western analysis and luciferase reporter assays. RESULTS: Exome sequencing revealed a novel somatic missense mutation Y665F in the STAT5b gene in two T-LGL patients diagnosed with WHO2009 criteria with a large CD8+ T-LGL clone (>90%). Only wild-type STAT5b was seen in the matched CD4+ control cells of these patients. Amplicon sequencing of exon 16 of STAT5b (corresponding to the Y665F site) in 158 T-LGL and 40 NK-LGL patients revealed an additional mutation (N642H) in one T-LGL and one NK-LGL patient, resulting in the 2% total frequency (4/198) of STAT5b mutations across all patients. The N642H and Y665F mutations were both located in the Src homology 2 (SH2) domain of STAT5b, which mediates dimerization and activation by trans-phosphotyrosine binding. STAT3 mutations previously reported in T-LGL patients were located in the corresponding domain. The transcriptional activity of wild-type and mutant STAT5b proteins (N642H and Y665F) was assayed in cells carrying a luciferase reporter with STAT5 binding elements. Luciferase activity of Hela cells transfected with the mutated STAT5b constructs was significantly increased compared to wild-type STAT5b. Furthermore, both the N642H and Y665F variants of STAT5b exhibited higher levels of tyrosine (Y694) phosphorylation than the wild type protein. The exon 21 in the SH2 domain of the STAT3 gene was also screened by ultra-deep next-gen amplicon sequencing, both from the original T-LGL patient cohort (n=77, patients with monoclonal disease) and 142 additional monoclonal/oligoclonal LGL patients. In the monoclonal cohort, a total of nine new STAT3 mutation positive patients were detected by amplicon sequencing, raising the total number of positive cases to 41 (53%) from the 32 identified by Sanger sequencing. Concomitant to the previously described Y640F, N647I, K658N, D661H, D661V and D661Y STAT3 mutations, several novel mutations in this gene were found: I659L, Q643H, G656C, K658H, K658R and D661I. In the oligoclonal LGL cohort, the mutation frequency was lower (31/142, 22%), suggesting that it may also include patients with reactive polyclonal LGL proliferation. CONCLUSIONS: Our mutational and functional data affirm that STAT family transcription factors play a critical role in the pathogenesis of LGL leukemia. In addition to the previously identified mutations in the STAT3 gene, we found recurrent somatic STAT5b mutations in LGL leukemia. Furthermore, our ultra-deep (10,000x) next-gen sequencing revealed small subclones of STAT3 mutations in patients with oligoclonal LGL. Both STAT3 and STAT5b mutations increased the phosphorylation and transcriptional activity of corresponding proteins. The detection of STAT mutations should be included in the diagnostic assessment of LGL leukemia. Disclosures: Koskela: Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Kallioniemi:TEKES-FiDiPro: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.