Normal T-cells Research Articles

SP600125 Attenuates Nicotine-Related Aortic Aneurysm Formation by Inhibiting Matrix Metalloproteinase Production and CC Chemokine-Mediated Macrophage Migration.

Nicotine, a major chemical component of cigarettes, plays a pivotal role in the development of abdominal aortic aneurysm (AAA). c-Jun N-terminal kinase (JNK) has been demonstrated to participate in elastase-induced AAA. This study aimed to elucidate whether the JNK inhibitor SP600125 can attenuate nicotine plus angiotensin II- (AngII-) induced AAA formation and to assess the underlying molecular mechanisms. SP600125 significantly attenuated nicotine plus AngII-induced AAA formation. The expression of matrix metalloproteinase- (MMP-) 2, MMP-9, monocyte chemoattractant protein- (MCP-) 1, and regulated-on-activation, normal T-cells expressed and secreted (RANTES) was significantly upregulated in aortic aneurysm lesions but inhibited by SP600125. In vitro, nicotine induced the expression of MCP-1 and RANTES in both RAW264.7 (mouse macrophage) and MOVAS (mouse vascular smooth muscle) cells in a dose-dependent manner; expression was upregulated by 0.5 ng/mL nicotine but strongly downregulated by 500 ng/mL nicotine. SP600125 attenuated the upregulation of MCP-1 and RANTES expression and subsequent macrophage migration. In conclusion, SP600125 attenuates nicotine plus AngII-induced AAA formation likely by inhibiting MMP-2, MMP-9, MCP-1, and RANTES. The expression of chemokines in MOVAS cells induced by nicotine has an effect on RAW264.7 migration, which is likely to contribute to the development of nicotine-related AAA.

Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5

Targeting mitochondrial respiration has emerged as an attractive therapeutic strategy in blood cancer due to their unique metabolic dependencies. In this study, we show that pyrvinium, a FDA-approved anthelmintic drug, selectively targets lymphoma T-cells though inhibition of mitochondrial functions and JAK2/STAT5. Pyrvinium induces apoptosis of malignant T-cell line Jurkat and primary T-cells from lymphoma patients while sparing T-cells from healthy donors. Increased level of active caspase-3 and decreased levels of Bcl-2 and Mcl-1 were also observed in Jurkat and lymphoma T-cells but not normal T-cells treated with pyrvinium. In addition, pyrvinium impairs mitochondrial functions by inhibit mitochondrial respiration, suppressing mitochondrial respiratory complex I activity, increasing ROS and decreasing ATP levels. However, the effects of pyrvinium were abolished in mitochondrial respiration-deficient Jurkat ρ0 cells, confirming that pyrvinium acts on lymphoma T-cells via targeting mitochondrial respiration. We further show that lymphoma T-cells derived from patients depend more on mitochondrial respiration than normal T-cells, and this explains the selective toxicity of pyrvinium in lymphoma versus normal T-cells. Finally, we demonstrate that pyrvinium also suppresses JAK2/STAT5 signaling pathway in Jurkat cells. Our study suggests that pyrvinium is a useful addition to T-cell lymphoma treatment, and emphasizes the potential therapeutic value of the differences in the mitochondrial characteristics between malignant and normal T-cells in blood cancer.

Drug-DNA Conjugated Gold Nanoparticles for the Treatment of Acute Myeloid Leukemia

INTRODUCTIONNovel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODSAuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTSConjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONSThis method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Disruption of ZEB1 Binding Causes Constitutive Activation of IL-15 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin-homing T-cells that represents 70-80% of all cutaneous lymphomas. Studies have shown that interleukin-15 (IL-15) plays a key role in T-cell oncogenesis by increasing T-cell survival, proliferation, migration and invasion in patient-derived CTCL cell lines. Previously, we have demonstrated that IL-15 is overexpressed in lesional biopsies and peripheral blood of CTCL patients (Mishra et al., Blood, 122:1826, 2013). However, the underlying mechanisms of IL-15 deregulation are largely unknown. Since epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T-cells from the blood of CTCL patients and performed a pyro-sequencing analysis to determine methylation of the IL-15 gene promoter. We observed significantly higher methylation in the IL-15 promoter of CD4+ T-cells from CTCL patients vs. normal donors (mean ± SEM of relative % methylation= 232.0 ± 49.18, n=9 vs 100.1 ± 7.619, n=6, P =0.03). Furthermore, methylation at the IL-15 promoter was higher in malignant CD4+ T-cells compared to non-malignant neutrophils from the same patient (mean ± SEM of relative % methylation = 232.0 ± 49.18 vs 52.31 ± 6.28%, N=9 each, paired t-test, P =0.0025). Using this approach, we also analyzed methylation of each 'CpG dinucleotide' in the IL-15 promoter. Once again, methylation was higher in CD4+ T-cells of patient versus normal donors and, within each patient, in CD4+ T-cells versus neutrophils. The CpG rich region of the human IL-15 promoter contains three putative binding sites for the known transcriptional repressor Zeb1, which has been reported to be mutated or deleted in CTCL. In order to examine the effect of CpG methylation at the Zeb1 binding region on IL-15 transcription, the CpG rich 5' regulatory region of the IL-15 promoter was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, the region of the IL-15 promoter containing the 3 ZEB1-binding sites is transcriptionally active in its native form (mean ± SEM of relative luciferase activity of promoter-less vector vs. IL-15 promoter vector = 77.00 ± 3.29 vs. 626.8 ± 25.05 respectively, N=3 each, P<0.0001). Deletion of the putative Zeb1 binding sites (BS#1, BS#2, BS#3 and BS#1-3) or the entire Zeb-binding region (BR) in the IL-15 promoter led to a significant increase in IL-15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL-15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1-3 vs. BR vectors = 99.56 ± 0.33 vs. 7135 ± 108.5 vs. 3940 ± 62.36 vs. 2372 ± 24.65 vs. 1099 ± 9.72 vs. 1525 ± 32.00, n=4 each; P <0.0001 each). Since CpG methylation of DNA can physically prevent transcription factor binding to regulatory regions of DNA, we hypothesized that CD4+ cells from CTCL patients might display reduced binding of Zeb1 to the IL-15 promoter thereby increasing IL-15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)-PCR, we observed a substantial loss of Zeb1 binding at the IL-15 promoter in the CD4+ cells of CTCL patients compared to those of normal donors (mean ± SEM of relative Zeb1 binding to IL-15 promoter in normal donor vs. CTCL patient CD4+ T-cells = 101.3 ± 1.48 vs. 25.31 ± 3.78, n=3 each; P <0.0001). Similarly, silencing Zeb1 in normal donor CD4+ T-cells caused an increase in IL-15 transcription. Finally, constitutive IL-15 overexpression was sufficient to cause a high penetrance lymphoproliferative disease in transgenic mice, with typical dermatologic features of human CTCL, including alopecia, scaly erythematous plaques/patches, and ulcerations, caused by an epidermotropic T-cell infiltrate. Thus, we defined Zeb1 as a novel repressor for IL-15 transcription in normal T-cells and show that epigenetic interference with its repressive function at the IL-15 promoter is associated with T-cell lymphoma in vivo, highlighting the role of epigenetic processes in inflammation-induced cancers and further supporting the role of Zeb1 as a putative tumor suppressor gene in CTCL DisclosuresPorcu:Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding.

Pathways for Oncogenesis in T-Cell Acute Lymphoblastic Leukemia Driven By DNA Methylation and Notch Signaling

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia predominantly found in children that has a worse prognosis than other forms of pediatric leukemia with few options for relapsed disease. These issues lead to a need for new targeted therapies. Many of the genes critical for normal T-cell differentiation are aberrantly expressed when patients develop T-ALL, and this knowledge can serve as the basis for rationale design of targeted therapies. In this list are DNA methyltransferase 3A (DNMT3A), a protein that is responsible for de novo DNA methylation, critical for the function of hematopoietic stem cells (HSCs), and NOTCH1, a protein involved in T-cell signaling and regulating cell fate decisions. Patients that with DNMT3A loss-of-functions mutations driving T-ALL have a worse prognosis than their counterparts with normal DNMT3A function. Previous attempts to directly target the action of NOTCH1 have not led to success, specifically gamma secretase inhibitors, so we aim to define the epigenetic regulation of this pathway and the co-operation between DNMT3A and NOTCH1 mutations may open the door for new therapies in patients with the poorest outcomes.We show here that ablation of Dnmt3a in murine HSCs using Mx1-Cre (Mx1-CRE:Dnmt3afl/fl) results in abnormal T-cell development, leading to an accumulation of progenitors at the double negative 2 (DN2) stage (CD4-, CD8-, c-Kit+, CD44+, CD25+) in a proportion of mice (but not all). In affected mice, although there is a significant accumulation of these progenitors, the downstream population of mature T-cells is essentially unchanged compared to wild-type mice. This developmental arrest is associated with increased expression of the Notch1 signaling pathway. Initially suspected as the mechanism given the native action of Dnmt3a, was differential DNA methylation of Notch1 and its downstream targets such as Hes1 and c-Myc, however, through performed bisulfite sequencing of the promoters for those genes and discovered that there was no difference in DNA methylation between wild-type and Dnmt3a-null thymic progenitor cells. The cellular difference underlying accumulation of this Dnmt3a-null thymic progenitor population appears to be increased resistance to apoptosis, and molecular studies are ongoing to explore the molecular mechanism behind this.The observations that thymic progenitor cells lacking Dnmt3a have modified expression of Notch1, and mutations of both DNMT3A and NOTCH1 have been found in T-ALL patient samples, indicates potential co-operation between these two pathways in T-ALL oncogenesis. To test the biological co-operation between these pathways in vivo, we introduced activated Notch1 intracellular domain (NICD) into wild-type and Dnmt3a-null hematopoietic progenitor cells and assessed the latency to T-ALL by bone marrow transplantation. Introducing NICD into a Dnmt3a-null background significantly accelerated T-ALL oncogenesis (median survival wild-type NICD = 72-days, median survival Dnmt3a-null NICD = 46-days, p < 0.001, Figure 1). To explore the molecular differences leading to enhanced T-ALL pathogenesis in a Dnmt3a-null background, we performed RNA-Seq on T-ALL blasts and found that murine leukemia samples with the combination of Dnmt3a loss-of-function and constitutive Notch1 activation show increased in Jak/Stat pathway signaling compared to T-ALL blasts with native Dnmt3a and normal double positive T-cells (CD8+, CD4+).We hypothesize that Jak/Stat pathway inhibitors could improve outcomes for T-ALL patients with combination DNMT3A loss-of-function and activating NOTCH1 mutations. Ruxolitinib is a well-known JAK1/JAK2 inhibitor used in the treatment of myelofibrosis where it has produced robust results. Given the alterations in Jak/Stat signaling, we plan to induce leukemia by elimination of Dnmt3a using the Cre based model, followed by viral introduction of the Notch1 mutant and then treat mice with ruxolitinib and compare survival. It is our hypothesis that the drug will increase latency to leukemia in this quick to develop murine model. Using developed drugs to target new pathways in T-ALL has the potential to offer new strategies to patients with difficult to treat relapsed disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Involvement of T-Cells in the Bone Pathology of Multiple Myeloma

Introduction: Multiple myeloma (MM) is a plasma cell dyscrasia that is heavily dependent on a permissive BM micro-environment. Components of the WNT pathway have been implicated in this process and specifically, WNT-inhibitors such as Dkk1, have been blamed for the osteolytic lesions of MM. WNT signaling is required for proper osteoblast development while the pathway is also involved in enhancing the self renewal potential of various stem cells. A source of WNT ligands in the BM are the T-cells; when stimulated by parathormone, T-cells secrete wnt10b that affects hematopoietic stem cell numbers. In the present study, we evaluated WNT signaling in the T-cells of patients with MM in order to test whether these bystander T-cells have an altered phenotype that could influence disease pathology.Methods: A total of 18 MM patients was analyzed after informed consent was obtained; the patients underwent BM biopsy for diagnostic and staging purposes. T-cells were isolated from BM mononuclear cells using magnetic beads with a purity of around 90%. Normal controls were T-cells isolated from healthy subjects. RNA was isolated and analyzed by RT-PCR for expression of PTH receptor (PTH1R), wnt10b and the canonical WNT signal transducer, b-catenin (bCAT).Results and discussion: BM concentration of T-cells was 15%, close to the normal values, indicating no major deviation from normal conditions. Activity of the WNT pathway was estimated by bCAT mRNA levels; relative to the control, bCAT levels in MM T-cells were uniformily suppressed. In a total of 8 evaluable samples, there was a 3x decrease in bCAT levels. Although WNT activity could be influenced by different factors, there is a possibility that it may be related to PTH activity and PTH receptor expression on patient T-cells. In all our cases, calcium was recorded within normal range and PTH values were also normal. However, PTH1R mRNA levels in MM T-cells were 14.2x lower compared to normal T-cells, a value that argues for a biological role in disease pathogenesis. A direct consequence of low PTH1R mRNA levels, according to our hypothesis, would lead to a decrease in WNT10b. Indeed, WNT10b levels were on average 90% of the normal with relative wide variation; in 3 samples, there was an average 3.2x increase while in 8, the levels were on average 21% of normal. On the clinical level, lower wnt10b activity, was more often associated with bone pathology while higher wnt10b activity was more often associated with extramedullary plasmacytomas. Our data show that T-cells may not be bystanders in the MM disease progression and certain manifestations sush as bone disease, could be associated with, aberrant T cell function. Ongoing studies are under way to link clinical and laboratory data. DisclosuresNo relevant conflicts of interest to declare.

Abstract B40: Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions.

Abstract Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK, but not all patients respond and complete responses are infrequent with single agent ibrutinib. We hypothesize that additional agents used in combination could enhance the breadth, depth and duration of responses. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy. Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 displayed synergistic cytotoxicity (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL cells from 9 patients, 5 displayed synergistic cytotoxicity and 4 did not, indicating substantial patient heterogeneity in response to the combination, presumably due to variations in genetic landscape. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors. Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy. In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned. Citation Format: Craig A. Portell, Mark J. Axelrod, Laura Kyle Brett, Vicki L. Gordon, Brian Capaldo, Jeffrey Xing, Stefan Bekiranov, Michael E. Williams, Michael J. Weber. Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B40.

STX11 functions as a novel tumor suppressor gene in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Their molecular pathogenesis has not been entirely elucidated. We previously showed that 6q24 is one of the most frequently deleted regions in primary thyroid T-cell lymphoma. In this study, we extended the analysis to other subtypes of PTCL and performed functional assays to identify the causative genes of PTCL that are located on 6q24. Genomic loss of 6q24 was observed in 14 of 232 (6%) PTCL cases. The genomic loss regions identified at 6q24 always involved only two known genes, STX11 and UTRN. The expression of STX11, but not UTRN, was substantially lower in PTCL than in normal T-cells. STX11 sequence analysis revealed mutations in two cases (one clinical sample and one T-cell line). We further analyzed the function of STX11 in 14 cell lines belonging to different lineages. STX11 expression only suppressed the proliferation of T-cell lines bearing genomic alterations at the STX11 locus. Interestingly, expression of a novel STX11 mutant (p.Arg78Cys) did not exert suppressive effects on the induced cell lines, suggesting that this mutant is a loss-of-function mutation. In addition, STX11-altered PTCL not otherwise specified cases were characterized by the presence of hemophagocytic syndrome (67% vs 8%, P = 0.04). They also tended to have a poor prognosis compared with those without STX11 alteration. These results suggest that STX11 plays an important role in the pathogenesis of PTCL and they may contribute to the future development of new drugs for the treatment of PTCL.

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Next-generation sequencing identifies deregulation of microRNAs involved in both innate and adaptive immune response in ALK+ ALCL.

Anaplastic large cell lymphoma (ALCL) is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK). We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS). In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL.

Release of cytokines in stored whole blood and red cell concentrate: Effect of leukoreduction.

Background:Storage time of blood components plays a major role in the accumulation of cytokines causing adverse transfusion reactions.Aims:The aim was to study the trend in the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha (TNF-α) and regulated upon activation, normal T-cells expressed and secreted (RANTES) during storage of whole blood (WB) and red cell concentrate (RCC) and to study the effect of leukoreduction (LR).Materials and Methods:WB sample was taken on 0, 7, 14, 21, and between 28 and 35 days and plasma aliquots were frozen. Samples from RCC and buffy-coat depleted RCC prepared using Optipress II were collected on 0, 7, 14, 21 and between 28 and 35 days. Cytokine estimation was done using ELISA development kits. Normal range of cytokines was established using 0 day samples of WB. Statistical analysis was done using nonparametric tests.Results and Conclusion:The normal range of IL-6 was 0-23 pg/ml, IL-8 0-12 pg/ml, TNF-α 0-3 pg/ml, and RANTES 1200-2000 pg/ml. IL-6 was in normal range and showed a decreasing trend during storage. IL-8 levels increased significantly from 0 to 35 days. In RCC, the highest level was 480 pg/ml on 28th day. It was in the normal range in buffy-coat depleted RCC up to 28 days. RANTES level was significantly low in buffy-coat depleted RCC compared to RCC. We conclude that WB has high levels of IL-8 and RANTES. The levels of cytokines are affected by storage period and LR. Comparison of WB and buffy-coat depleted RCC shows significantly low levels of IL-6, IL-8, and RANTES in buffy-coat depleted RCC. This study emphasizes the use of red cell components instead of WB and buffy-coat depleted RCC instead of RCC.

Synergistic Cytotoxicity of Ibrutinib and the BCL2 Antagonist, ABT-199(GDC-0199) in Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL): Molecular Analysis Reveals Mechanisms of Target Interactions

Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK. In Phase II studies of single-agent ibrutinib in MCL (Wang ML et al, NEJM 2013) and CLL (Byrd JC, et al, NEJM 2013) the overall response rate was 68% and 89% (CR, PR, and PR with lymphocytosis), respectively, with PR as the best response in the majority of patients. Thus, not all patients respond and complete responses are infrequent with single agent ibrutinib. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines using a focused drug panel (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy.Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 substantially induced apoptosis compared to each single agent alone (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL and normal donor samples, heterogeneity in response to the combination of ibrutinib and ABT-199 was seen. When evaluated by Bliss modeling, 5 of 9 CLL samples had a synergistic improvement in apoptosis with the combination with the other 4 having no change. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors.Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy.In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned. DisclosuresOff Label Use: Pre-clinical data with ABT-199 for MCL and CLL, not FDA approved. Williams:Pharmacyclics, Janssen: Consultancy, Research Funding.

The NOTCH1 Driven Long Non-Coding RNA Repertoire in T-Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer resulting from the malignant transformation of T-cell precursors. Genetic studies in T-cell acute lymphoblastic leukemia (T-ALL) have uncovered a remarkable heterogeneity of genetic defects. Amongst these, NOTCH1activating mutations are the most frequently occurring events (>50%) in T-ALL. Since long non-coding RNAs (lncRNAs) are emerging as important players in oncogenesis, we decided to decode the NOTCH1 driven lncRNA transcriptional landscape in T-ALL and normal T-cell development.Methods and Results: RNA-sequencing was performed following pharmacological inhibition (GSI) of the NOTCH1 mutant and gamma secretase inhibitor (GSI) sensitive T-ALL cell line CUTLL1 in a time series experiment as well as for human CD34+ thymic progenitor T-cells cultured on an OP9 feeder layer with or without DL1-triggered NOTCH1 stimulation. First, we validated both model systems by confirming robust regulation of multiple canonical known protein coding NOTCH1 target genes including DTX1, NOTCH3 and NRARP. Next, we identified distinct subsets of NOTCH1 regulated lncRNAs in both experiments with an overlap of 27 commonly regulated NOTCH1 driven and previously annotated lncRNAs. An even larger number of novel, unannotated T-ALL/T-cell specific lncRNAs was found to be NOTCH1 regulated. Next, we took advantage of publically available ChIP-sequencing data for ICN1 and enhancer specific chromatin marks in CUTLL1 (Wang et al., PNAS, 2013), allowing the selection for direct regulated lncRNAs with enhancer properties. Amongst these lncRNAs, the recently described LUNAR1 lncRNA (Trimarchi et al., Cell, 2014) was present as top candidate in our dataset, thus validating this approach for further selecting bona fide NOTCH1 regulated lncRNAs. In a first step towards functional annotation of this subset of selected lncRNAs, we performed so-called guilt-by-association analysis through correlating expression levels of the individual lncRNAs with transcriptome data for all protein coding genes followed by gene set enrichment analysis in a large cohort of primary T-ALL patients. Subsequent enrichment mapping of significant gene sets yielded markedly different gene set clustering patterns for each of the individual analyzed lncRNAs, as well as distinct annotated related functionalities such as cytokine signaling, TCA-cycle, DNA replication and repair and translation. Prioritarization of lncRNAs for further functional validation was performed by measuring their expression in an extended panel of GSI-treated T-ALL cell lines (HPB-ALL, DND-41, T-ALL1 and ALL-SIL), sorted subsets of CD34+ and CD4+/CD8+ double positive thymocytes and an independent T-ALL patient cohort.Conclusion: We present the landscaping of an integrated lncRNA network acting downstream of NOTCH1 signaling in T-ALL and normal T-cells. These data pave the way towards the development of novel therapeutic strategies impacting on hyperactive NOTCH1 signaling. DisclosuresNo relevant conflicts of interest to declare.

Identification of a New Subclass of ALK Negative Anaplastic Large Cell Lymphoma Expressing Aberrant Levels of ERBB4 Transcripts

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.To discover biomarkers and/or genes potentially involved in ALK- ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a large gene expression profiling (GEP) data set including 249 cases of T-NHLs and normal T-cells. Among the top outliers, ERBB4 and COL29A1 genes were exclusively expressed in a subset of ALK- ALCL cases, and resulted highly correlated. Differential analysis comparing the expression profiles of ERBB4/COL29A1+ to ALK+ ALCL patients identified 48 genes up- and 37 down-regulated (qval < 0.0076). The most significantly over-represented functional category included transcriptional regulators. Gene-set-enrichment-analysis (GSEA) indicated that ERBB4/COL29A1+ samples shared activation of specific pathways such as inflammatory response, TNF-NF-κB, JAK-STAT, cytokine, and angiogenesis. RNA sequencing and 5’RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two ERBB4 truncated transcripts (referred as I20ΔERBB4 and I12ΔERBB4), displaying Transcription Starting Sites (TSS) in intron 12 and 20, respectively. On the contrary, full length COL29A1 mRNA was expressed in ERBB4/COL29A1+ samples. RT-qPCR analysis performed on 170 T-NHL samples (51 PCTL-NOS, 44 ALK+ ALCL, and 75 ALK- ALCL) highlighted specific expression of ERBB4 aberrant transcripts in 25% of ALK- ALCL (18 out of 75). ERBB4+ patients showed higher expression of I20ΔERBB4 as compared to I12ΔERBB4 transcript. ERBB4 expression at protein level was confirmed by immunohistochemistry and Western Blotting on selected cases. Inspection of intronic regions spanning two hypothetical TSS revealed the presence of Human Endogenous Retrovirus (HERV) Long Terminal Repeats (LTR) displaying promoter activity, as demonstrated by luciferase assays.In conclusion, we defined a new subclass of ALK- ALCL characterized by ectopic expression of ERBB4 and COL29A1 genes. To the best of our knowledge, ERBB4 truncated transcripts carrying intronic 5’UTR have never been described before. Further studies are required to address whether the expression of ERBB4 aberrant transcripts may contribute to the ALCL transformation and lymphoma maintenance. This information might lead to more rational therapeutic approaches for ERBB4+ ALCL patients. DisclosuresNo relevant conflicts of interest to declare.

STX11 Acts As a Novel Tumor Suppressor Gene in Peripheral T-Cell Lymphomas

Introduction: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas noted for their poor prognosis. Their molecular pathogenesis has not been entirely elucidated. We previously found that primary thyroid T-cell lymphoma (PTTL) is a distinct entity among heterogeneous PTCLs and that this disease is characterized by the genomic loss of 6q24 (Br J Haematol., 161:214-223). In this study, we extended the analysis to other types of PTCLs and performed functional assays to identify causative genes located on 6q24.Methods: Focusing on chromosome 6q loss, we reexamined previous comparative genomic hybridization data from 267 PTCL cases comprising 6 PTTLs, 51 PTCLs-not otherwise specified (NOS), 62 adult T-cell leukemias/lymphomas, 35 natural killer (NK)-cell lymphomas, 39 angioimmunoblastic T-cell lymphomas (Genes Chromosomes Cancer, 46:37-44), and 74 anaplastic large cell lymphomas (Br J Haematol., 140:516-526). Gene expression levels were determined by using published gene expression profiling (GEP) data (GSE6338 and GSE19069) and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequently, we established Tet-Off cell lines belonging to several lineages (6 T-cell lines, 1 NK-cell line, 4 B-cell lines, 1 myeloid cell line, and 3 epithelial cell lines) for functional analyses.Results: Genomic loss of 6q24 was observed in 8% (n = 267) of PTCL cases, and it occurred most frequently in PTTL cases (67%; n = 6). All the genomic losses were heterozygous; homozygous loss of this region was not observed in our analysis. The smallest region of deletion, observed in a PTTL case, was considered the minimal common region (MCR) of 6q24 loss. The MCR contained 2 known coding genes, STX11 and UTRN. Combined GEP data and quantitative RT-PCR analyses showed that the expression of STX11, but not UTRN, was markedly lower in PTCL than in normal T-cells. We therefore regarded STX11 as the most probable candidate gene located in 6q24. Syntaxin 11, encoded by STX11, is a t-SNARE protein that plays a role in binding vesicles to cell membranes, and alteration of STX11 in the germline causes familial hemophagocytic syndrome type 4.To further evaluate genomic alteration of STX11, mutation analysis was performed on PTCL-NOS and PTTL cases as well as T-cell lines, for which adequate DNA was available. This revealed STX11 mutations in 2 cases (1 PTCL-NOS case and 1 T-cell line). Wild-type STX11 expression suppressed the proliferation of T-cell lines bearing genomic alterations at the STX11 locus only, and it did not show suppressive effects on other lineage cell lines (Fig. 1). Expression of STX11 induced cellular apoptosis in the cell line, although the number of apoptotic cells induced was relatively small. Interestingly, expression of a novel STX11 mutant (p.Arg78Cys), observed in a T-cell line, did not exert suppressive effects on the induced cell lines suggesting that there was a loss-of-function mutation (Fig. 2). Finally, we evaluated the clinical impact of STX11 alteration in PTTL and PTCL-NOS cases where data were available. This showed that PTCL-NOS cases with genomic alterations of STX11 tended to have a poorer prognosis than those without (Fig. 3; P = 0.069).Conclusion:In the present study, we examined the MCR of 6q24 loss and showed that STX11 acts as a tumor suppressor gene in PTCLs only. These findings provide a novel approach for understanding the molecular pathogenesis of PTCLs, and they may contribute to the future development of new drugs for the treatment of PTCLs. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microrna-374b Suppresses Proliferation and Promotes Apoptosis in T-Cell Lymphoblastic Lymphoma By Repressing Wnt-16 and AKT1 and Is a Promising Predictive Biomarker for Patients Survival

Background: De-regulation of microRNA (miRNA) has been extensively investigated in both Hodgkin (HL) and non-Hodgkin lymphomas (NHL), however, little is known about the roles of miRNAs in T-cell lymphoblastic lymphoma (T-LBL). To understand the involvement of miRNAs in the development and treatment of (T-LBL), miRNA profiles were compared between tumor and corresponding non-tumor tissues.Methods: miRCURY LNA array was used to generate miRNA expressing profile. Real-time quantitative PCR and immunohistochemistry (IHC) were applied to detect the expression of miR-374b, AKT1 and Wnt16 in T-LBL samples. Dual-luciferase reporter assay was conducted to confirm target associations of miR-374b. The tumor suppressive effect of miR-374b was determined by both in-vitro and in-vivo studies.Results: The expression of 380 miRNAs was evaluated in five human T-LBL tissues and correspondence normal peripheral blood T-cells by microRNA microarrays. The down-regulation of miR-374b was frequently detected in primary T-LBL tissues, which was significantly associated with patients worse overall survival (P<0.001) and increasing risk of recurrence (P=0.012). Functional assays demonstrated that miR-374b could suppress T-NHL cells proliferation in vitro and in vivo. Furthermore, miR-374b could also sensitize cells to both serum starvation- and chemotherapeutic agent-induced apoptosis. Significantly, subsequent investigation characterized two AKT pathway associated molecules, AKT1 and Wnt16, as direct targets of miR-374b and account for the effect of miR-374b in T-NHL cells. Consistently, in tissues of our T-LBL patients, AKT1 and Wnt16 expression was inversely correlated with miR-374b levels, and could also be independent predictors of recurrence and survival of these patients.Conclusions: Our data highlight the molecular etiology and clinical significance of miR-374b in T-LBL. Targeting miR-374b may represent a new therapeutic strategy to improve the therapy effect and survival for T-LBL patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Recurrent T-Cell Receptor γ Rearrangement of the Vγ9-JγP Type Represents the Major Clone of γδ T-Cells in Normal Individuals and Is Highly Suppressed in Lymphoproliferative Disorders

INTRODUCTIONγδ T-cells expressing T-cell receptor (TCR) type Vγ9Vδ2 are well-established mediators of anti-tumor immunity and have demonstrable HLA-independent cytotoxic activity against both B- and T-cell non-Hodgkin lymphoma cells. Initial clinical trials using ex vivo expanded, adoptively transferred γδ T-cells to enhance tumor immunity have shown some promise but overall results have been lackluster. This indicates that wholesale polyclonal expansion prior to transfer is not likely to be effective. Thus more refined approaches are necessary. This requires a better understanding of their TCR repertoire. We recently developed a next generation sequencing (NGS)-based method for evaluating the spectrum T-cell receptor gamma (TRG) gene rearrangements present in clinical samples. In an effort to better understand the TCR repertoire of γδ T-cells we used this strategy to evaluate a series of samples from normal individuals and from individuals with B and T-cell lymphoproliferative disorders (LPDs).METHODSDNA was isolated from samples from 11 normal individuals (all peripheral blood, PB), 11 patients with a T-cell LPD (6 PB, 3 bone marrow; BM, 2 FFPE tissues), and 5 patients with a B-cell LPD (Hairy cell leukemia; HCL, 4 PB, 1 BM). γδ T-cells were sorted from the PB of 4 of the healthy donors by FACS. TRG rearrangements were PCR amplified using consensus primers and NGS libraries were prepared and sequenced on the Ion Torrent PGM platform. The data was analyzed as follows. NGS typically yielded up to 400,000 sequencing reads which were grouped by identical V, J, and CDR3 sequences into unique rearrangements (typically 15-30,000). The prevalence of a particular TRG rearrangement (or CDR3 sequence) was determined by the number of individual NGS reads with this unique sequence per the entire data set (percent of total reads). All rearrangements were then ranked by their prevalence.RESULTSWe sequenced the TRG repertoire of isolated γδ T-cells from the peripheral blood of normal individuals (n=4). We found that a recurrent Vγ9-JγP rearrangement with the CDR3 sequence CALWEVQELGKKIKVF was always (4 of 4 samples) the most prevalent rearrangement in normal γδ T-cells (3.2-11.7-fold more prevalent than the second most common rearrangement; representing 4-11.9% of total reads). Similarly, analysis of a larger set of unsorted normal peripheral blood samples demonstrated high prevalence of the same canonical CDR3 in 5 out of 7 samples, confirming that it is very common in most individuals relative to all TRG rearrangements (among the top 10 most prevalent CDR3s in 4 of 5 samples). We also sequenced the TRG repertoire in 11 samples from patients demonstrating evidence of involvement by a T-cell LPD. Unexpectedly, all Vγ9-JγP type rearrangements were strongly suppressed in these samples including the one with the canonical CDR3. The canonical rearrangement (present in 6 /11 cases), or the most abundant Vγ9-JγP rearrangement when the canonical rearrangement was absent (absent in 5/11 cases), represented on average 0.036 ±0.024 % of all NGS reads in the samples from patients with T-cell LPDs compared to 0.72 ±0.72 % of total NGS reads in the normal controls. This represents on average a 19.9 fold reduction in the T-cell LPD samples. The median rank by abundance of the top Vγ9-JγP rearrangement dropped from 9th (normals) to 291st (T-cell LPD cases) indicating that the suppression was not simply a consequence of the presence of an abundant malignant T-cell clone in the data. The overall distribution of TRG V-segment usage in the normal and neoplastic samples was comparable (Vγ9: 9.9 ±0.55 % and 7.2 ±2.79 %, respectively). In 2 samples from patients with HCL, Vγ9-JγP rearrangements were reduced to a similar extent to that seen in the T-cell LPD cases (9.5-fold reduced; average 0.06% of NGS reads; average rank order 451st). In the other 3 cases of HCL the findings were very similar to those seen in the normal samples.CONCLUSIONSWe identified a recurrent Vγ9-JγP rearrangement by NGS representing the most abundant CDR3 in sorted γδ T-cells from normal individuals. This population, along with other clones with Vγ9 rearrangements, appeared specifically suppressed in all samples from patients with T-cell LPDs and in 2 or 5 samples from patients with B-cell LPDs (HCL), perhaps indicating a role in the disease process. Additional samples from a wider range of T- and B-cell LPDs are being analyzed. DisclosuresNo relevant conflicts of interest to declare.

Downregulating Notch Signaling in KrasG12D/+ Mice Inhibits Both T-Cell Leukemia and Myeloproliferative Neoplasm in a Cell-Autonomous Manner

Notch signaling is implicated in diverse functions in hematopoiesis, including stem cell maintenance, cell fate specification, cell proliferation, and apoptosis. Aberrant Notch signaling is associated with the pathogenesis of various hematopoietic malignancies. NOTCH1 mutations that lead to elevated intracellular Notch1 activity are identified in 50-60% of patients with acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and in 100% of oncogenic Ras-induced T-ALL in mice. Although Notch1 mutations contribute to the malignant transformation of normal T-cells to T-cell leukemia/lymphoma initiating cells in the KrasG12D/+-induced T-ALL model, it remains unclear whether Notch1 signaling is necessary for T-ALL genesis. Moreover, it remains controversial whether loss of Notch signaling promotes myeloproliferative neoplasms (MPN) in a cell-autonomous manner.To address these questions, we used genetic approaches to downregulate Notch signaling in KrasG12D/+ mice, which develop both T-ALL and MPN. Downregulation of Notch signaling in hematopoietic cells is achieved through Mx1-Cre–mediated conditional expression of Rosa26-GFP-dnMAML1, which inhibits canonical Notch signaling, or conditional knockout of Pofut1, which catalyzes O-fucosylation of Notch receptors and modulates Notch receptor ligand interactions. We found that either overexpression of dnMAML1 or deletion of Pofut1 significantly enhanced MPN phenotypes and shortened the survival of KrasG12D/+ mice. However, several pieces of evidence suggest that downregulation of Notch signaling in non-hematopoietic cells might influence MPN development. First, 100% of compound mice developed atopic dermatitis-like disease that is shown to promote MPN in a cell non-autonomous manner. Second, ~30% of endothelial cells were GFP-positive (expressing dnMAML1) in KrasG12D/+; Rosa26GFP-dnMAML1/+ mice.To determine whether downregulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of KrasG12D/+, KrasG12D/+; Rosa26GFP-dnMAML1/+, or KrasG12D/+; Pofut1-/- bone marrow cells (CD45.2+) along with congeneic competitor cells (CD45.1+) into lethally irradiated mice (CD45.1+). As expected, inhibiting Notch signaling significantly blocked T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras and preserved intact Notch signaling. Surprisingly, we found that the percentage of donor-derived myeloid cells was significantly lower in recipients transplanted with KrasG12D/+; Rosa26GFP-dnMAML1/+ or KrasG12D/+; Pofut1-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with KrasG12D/+ cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in KrasG12D/+; Rosa26GFP-dnMAML1/+ bone marrow than that in KrasG12D/+ bone marrow, we investigated whether the absence of the MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of KrasG12D/+ or KrasG12D/+; Rosa26GFP-dnMAML1/+ splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 12 recipient mice with KrasG12D/+; Rosa26GFP-dnMAML1/+ cells developed a donor-derived (KrasG12D/+; Rosa26GFP-dnMAML1/+) MPN disease, while 6 out of 12 recipient mice with KrasG12D/+ cells died with donor-derived MPN (P=0.02). Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner. We are currently working on the underlying mechnisms. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia.

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3'untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

Вплив імунокорекції на запальний процес урогенітального тракту у чоловіків з безпліддям

We studied the concentration of metalloproteinases, pro-and anti-inflammatory cytokines, lymphocyte activation markers in seminal plasma of men with oligosymptomatic forms of chronic inflammation of the urogenital tract complicated with infertility. It was shown that chronic inflammation of the urogenital tract shifts the levels of cytokine profile, reduces the concentration of metalloproteinase-2 (576.4 +/- 89.3 ng/ml, P < 0.001), chemokines - fractalkine (16.3 +/- 1.3 pg/ml, P < 0.001) and chemmmokine regulated by activation, expression and secretion of normal T-cells (RANTES (12.7 +/- 1.0 pg/ ml, P < 0.001). Additionally, we observed a sharp increase in IL-8 (215.5 +/- 7.8 pg/ml, P < 0.01), MCP-1 (926.4 +/- 30.2 pg/ml, P < 0.001) and elevation of the CD25 + / CD95 + ratio. These observations point for alterations in apoptotic mechanisms in pathological forms of the generative cells and their accumulation in the sperm. Prolonged inflammation of the genital area is accompanied by depletion of the local immune system resulting in the development of infertility. The multidirectional shifts in IL-10/IL-12 index have been established: an increase in the group of patients with elevated level of hypercapitated spermatozoa and its reduction in microsomatic morphotype of spermatozoa. These data emphasize the importance of the microenvironment in maturation of gametes. It was shown that an inclusion of alfagin and gepatomax in immunorehabilitation of men with oligosymptomatic forms of urogenital infections improves leukocyte subpopulation content of sperm and concentrations of pro-and anti-inflammatory cytokines, and therefore, results in an increase in fertilizing potential.